Method for preventing and treating renal disease

ABSTRACT

Assays, methods and kits for predicting a subjects (e.g., human) risk of primary glomerulopathy, secondary glomerulopathy or recurrence (e.g., post-transplant recurrence) of any glomerular disease include examining cells for the presence or absence of cytoskeletal disruptions or rearrangements and examining cells for modulation of expression and/or activity of markers such as SMPDL-3b. Assays for predicting if a diabetic subject will develop kidney disease or a patient with FSGS will develop recurrent disease after transplant also include examining cells for the presence or absence of cytoskeletal disruptions or rearrangements and examining cells for modulation of expression and/or activity of markers such as SMPDL-3b. Also described herein are compositions and methods for treating and preventing the aforementioned disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. Divisional Application of Ser. No. 13/879,892filed Apr. 17, 2013, which is a 371 National Stage Application ofInternational Application No. PCT/US11/56272, filed Oct. 14, 2011, whichclaims priority to U.S. Provisional Application No. 61/481,485, filedMay 2, 2011, and U.S. Provisional Application No. 61/394,532, filed Oct.19, 2010, the entireties of which are incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates generally to the fields of cellular biology,molecular biology, nephrology, medicine and transplantation.

BACKGROUND

Presently, there is no predictive test for the development of glomerulardisorders, and diagnosis is based on kidney biopsies, a very invasivediagnostic procedure. As an example, there is no predictive test forrecurrent focal segmental glomerulosclerosis (FSGS) after a kidneytransplant. FSGS accounts for up to 20% of end-stage renal disease(ESRD) and is the most common progressive glomerular disorder affectingthe pediatric population. Although renal transplantation remains thebest treatment option for patients with FSGS reaching ESRD, recurrentFSGS after transplantation occurs in 30-70% of the patients and markedlyaffects graft survival. The ability to predict which patients are athigh risk for recurrent disease remains a challenge. As a secondexample, although 20-40% of patients with diabetes will develop diabeticnephropathy (DN), the identification of those patients at risk remainselusive.

The ability to predict the development of proteinuria and glomerulardisorder in any primary or systemic illnesses that can affect the kidneyis elusive (e.g., lupus, HIV, hepatitis, hemathological disorders,sarcoidosis). Similarly, no methodology exists for predicting if familymembers of patients with a non-genetic proteinuric glomerular disorderare at risk to develop the disorder. A prediction assay is also lackingfor patients undergoing a kidney transplant to determine the risk forthe development of kidney disease after kidney transplant (e.g.,transplant glomerulopathy, rejection, allograft nephropathy, recurrenceof the primary disease). Furthermore, for any renal (kidney) diseaseand/or glomerulopathy that can be either idiopatic (e.g., Membranous,minimal change, IgA, Membranoproliferative, FSGS, paucimmuneglomerulonephritis), genetic (e.g., FSGS, storage disorders, Alport's)or secondary to a systemic illness (e.g., lupus, diabetes, HIV,hepatitis, hemathological disorders, sarcoidosis, other autoimmunedisorders), the ability to predict what are the better treatmentstrategies for a specific patient is unrealized.

Proteinuria, kidney injury, renal failure and renal-related conditionscontribute significantly to morbidity and mortality of affectedpatients. Proteinuric renal failure and/or kidney injury is often theresult of local or systemic illnesses that affect the function of keyelements of the glomerular filtration barrier of the kidney, a complexcellular structure that under physiological conditions prevents theleakage of protein from the blood side to the urinary side¹. Althoughglomerular disease is the most common cause of pathological proteinuria,proteinuria can also be the result of tubular dysfunction or proteinoverflow. Among the three major components of the glomerular filtrationbarrier (endothelial cells, glomerular basement membrane and podocytes),the podocyte is a highly specialized cell that is usually affected inthe early phases of proteinuric glomerular disorder as a result ofdiverse insults (e.g., systemic illnesses, autoimmune diseases,infectious diseases, toxic agents, drugs and others). These insultscause podocyte actin cytoskeleton remodeling and lead to clinicallyrelevant proteinuria. Clinically relevant proteinuria is generallydefined as urinary excretion of more than 150 mg of protein per 24 h or150 mg/g creatinine on a spot urine sample or as urinary excretion ofalbumin of more than 30 mg per 24 h or 30 mg/g creatinine on a spoturine sample. Podocyte injury is also an important feature of DN, whichis the most common cause of end stage renal disease in the UnitedStates. As proteinuric chronic kidney disease represents a growingepidemic despite available treatments, there is a need for thedevelopment of new drugs or of new indications for existing moleculesand compounds.

SUMMARY

Assays, methods and kits for predicting a subject's (e.g., human) riskof primary glomerulopathy, secondary glomerulopathy or post-transplantrecurrence of any glomerular disease (e.g., FSGS, DN, Membranousnephropathy, minimal change disease, IgA nephropathy,Membranoproliferative glomerulopathy, lupus nephritis, myeloma kidney,hypertensive nephrosclerosis, paucimmune glomerulonephritis,preeclampsia, amyloidosis, cryoglobulinemia, thrombotic thrombocytopenicpurpura, Hemolytic uremic syndrome, scleroderma kidney, Alport'sglomerulopathy, transplant glomerulopathy storage disorders and otherrare genetic disorders) are described herein. Assays and methodsdescribed herein may be used to predict the development of glomerularrenal disorders (e.g., development of albuminuria, proteinuria, clinicalor histological evidence of any of the above conditions). Assaysdescribed herein may also allow for the identification of a therapeuticagent for preventing or treating glomerular diseases (e.g., FSGS, DN,Membranous nephropathy, minimal change disease, IgA nephropathy,Membranoproliferative glomerulopathy, lupus nephritis, myeloma kidney,hypertensive nephro sclerosis, paucimmune glomerulonephritis,preeclampsia, amyloidosis, cryoglobulinemia, thrombotic thrombocytopenicpurpura, Hemolytic uremic syndrome, scleroderma kidney, Alport'sglomerulopathy, transplant glomerulopathy, storage disorders and otherrare genetic disorders). Such assays include high throughput screeningassays (e.g., high throughput screening of libraries of compounds).Identification of patients at risk for kidney disease and in vitrodetermination of specific existing and new drugs to be utilized for eachindividual can be achieved using the assays and methods describedherein, providing for the development of a personalized nephrologyapproach to diagnosis and treatment that may, for example, replacekidney biopsies. A specific kit for each glomerular and/or renaldisorder can be provided in combination with a disease-specific set ofdrugs to be tested in vitro to predict a subject's response totreatment. As shown in FIGS. 1A, 1B, 1C, 1D, 1E, 2A, 2B, 2C, 2D, 2E, 2F,2G, 3A, 3B, 3C, 3D, 3E, 3F, 4A, 4B and 4C, the assays, methods and kitsdescribed herein provide for the utilization of existing and approveddrugs (e.g., drugs for off label use) with a personalized approach.

In one embodiment, the assays, methods and kits involve cultured humanpodocytes that display a disease-specific phenotype when contacted witha biological sample from a subject having or predisposed to the specificdisease (e.g., FSGS, DN, etc.). In the case of FSGS, the presence of asoluble permeability factor (PF) in the serum of patients with high riskfor recurrence (e.g., after kidney transplantation) has been shown: thepresence of a PF above a preset range of activity is associated with arecurrence rate as high as 86%. However, the specific nature andfunction of PF remains unclear; even less clear is the existence ofspecific podocyte targets for recurrent disease, leading to podocytemalfunction and detachment, similar to what has been described inprimary FSGS. In the experiments described herein, a translationalapproach to cell culture, where target cells of interest are studiedafter exposure to the sera of patients with a given disorder (instead ofthe traditional fetal bovine serum), has been applied to normal humanpodocytes cultured in the presence of the sera of 18 patients with FSGScollected immediately before transplantation and initiation ofimmunosuppression. Based on the actin cytoskeleton rearrangement thatwas observed by confocal microscopy, a quantitative assessment of adiseased phenotype has been established by counting the number of cellswith disruption of stress fibers (the hallmark of a diseased phenotypefor podocytes) among 100 cells being evaluated in adjacent fields (FIGS.1A-1E). Further, a correlation between the modulation of SMPDL-3b andthe degree of cell malfunction has been developed (Fornoni et al, SciTransl Med vol. 3:85ra46, 2011), offering a bioassay that includes amore objective and quantitative assessment of risk (FIGS. 2A-2G). Thedevelopment of a SMPDL-3b reporter podocyte or other transfectablemammalian cell line (e.g., HEK cell line) that allows for a directcorrelation between a marker (e.g., luciferase activity) detected afterexposure to patient sera in the presence or absence of a given drug andclinical outcome will offer a fast and more quantitative methodpredictive of clinical outcome. In this bioassay, data can be confirmedby Western blot and PCR as reported (Fornoni et al, Sci Transl Med vol.3:85ra46, 2011), for example. As SMPDL-3b affects cellular metabolism,function, and survival, luciferase activity in cells exposed to patientsera are studied alone or in conjunction with determination of cellularlipid content, apoptosis, oxygen consumption rate and quantitativemeasures of oxidative stress (e.g., as determined by seahorsetechnology). The assays, methods and kits described herein provide asensitive method to identify, prior to transplantation, those patientsthat are at risk for the development of recurrent FSGS or otherglomerular disease, which in turn will affect the choice of therapeuticagents to be administered at time of transplantation to more or lessaggressively prevent recurrent disease.

The experiments described herein additionally show that cellenlargement, blebbing and loss of stress fibers, lipid accumulation,modulation of SMPDL-3b and apoptosis are observed in normal podocytesexposed to the sera of diabetic patients with DN, but not in podocytesexposed to sera from normal non-diabetic controls or from diabeticpatients without DN. Thus, the assays, kits and methods described hereinprovide for the screening of patients with diabetes for their risk todevelop DN (FIGS. 3A and 3B). Advantageously, a specific kit for eachglomerular disorder is provided in combination with a disease-specificset of drugs to be tested in vitro to predict a subject's response totreatment.

Methods for lowering plasma membrane and cellular cholesterol/lipids forthe prevention, treatment, cure, or reversal of renal-related disordersby the use of cyclodextrin (CD), its derivatives or any other drugcapable of lowering plasma membrane and cellular cholesterol/lipids, arealso described herein. Strategies that reduce cellularcholesterol/lipids and that do not solely affect the cholesterolsynthetic pathway (such as statins), but also the influx and effluxmechanisms leading to cholesterol/lipids accumulation, can be utilizedfor the prevention and cure of renal-related disorders. CD, itsderivatives or any cellular cholesterol-lowering agent belonging to aclass of compounds other than statins (such as chromium picolinate) areencompassed by the compositions and methods described herein. Suchstrategies also include the modulation of sphingolipid-related enzymes,as it was demonstrated in the experiments described below thatsphingolipid-related proteins can modulate the cellular cholesterolcontent (FIGS. 5A and 5B). As accumulation of cellularcholesterol/lipids occurs with diabetes, aging, obesity and otherchronic inflammatory diseases, and visceral cholesterol accumulationresults in organ malfunction, cyclodextrin derivatives can be morebroadly utilized for the prevention and the cure of these other medicalconditions. Therefore, CD, its derivatives or any other drug that lowersplasma membrane or cellular accumulation of cholesterol/lipids mayresult in the prevention and cure for obesity and diabetes and may slowaging. In the example shown in FIGS. 6A and 6B, the following set ofdata was generated. Cholesterol depletion by intravenous injection ofHydroxypropyl-beta-cyclodextrin (CD) protects from Lipopolysaccharide(LPS) induced proteinuria (FIG. 6A) in mice and from LPS-inducedintracellular signaling mediated by MyD88 in isolated glomeruli (FIG.6B). Cholesterol depletion with Methyl-beta-cyclodextrin (CD) preventsseveral phenotypic changes observed in normal human podocytes exposed tothe sera of patients with diabetic nephropathy. Those phenotypic changesinclude: cell blebbing, lipid accumulation, and cell apoptosis (FIGS.3A-3F). As modulation of SMPDL-3b expression in podocytes leads to amodulation of lipids and lipid related proteins (such as ASMase, FIGS.5A and 5B), modulation of SMPDL-3b results in a modulation of cellularlipid content that renders cells more or less susceptible to injury(FIGS. 5A and 5B).

Described herein is an assay for determining if at least one subject whois at risk for a primary or secondary proteinuric glomerular disorder orwho has a primary or secondary proteinuric glomerular disorder is atrisk for primary or secondary disease development, progression orrecurrence of the proteinuric glomerular disorder after kidneytransplantation. The assay includes: contacting a biological sample(e.g., blood, saliva, serum, plasma, tissue, and urine) from the atleast one subject with a culture of human podocytes; examining thepodocytes for the presence or absence of cytoskeletal disruptions orrearrangements resulting in determination of a phenotype of thepodocytes; and correlating the presence of cytoskeletal disruptions orrearrangements with an increased risk of development, progression orrecurrence of the proteinuric glomerular disorder after kidneytransplantation in the subject. The proteinuric glomerular disorder(e.g., FSGS or diabetic nephropathy) is typically a primary proteinuricglomerular disorder, a secondary proteinuric glomerular disorder, or apost-transplant proteinuric glomerular disorder. The at least onesubject can be a plurality (e.g., 2, 3, 4, 5, 10, 15, 20, 50, 100) ofsubjects who are at risk for a primary or secondary proteinuricglomerular disorder or who have a primary or secondary proteinuricglomerular disorder. The cytoskeletal disruptions or rearrangements canbe examined by, for example, microscopy, Western blotting, ELISA, flowcytometry and reporter assays. In the assay, the phenotype can becompared to a negative control and a positive control. The assay canfurther include analyzing the phenotype quantitatively based on astandard curve generated with a dose-dependent chemical disruption ofactin cytoskeleton. By using the assay, for the at least one subject whois at risk for a primary, secondary or recurrent proteinuric glomerulardisorder or who has a primary, secondary or recurrent proteinuricglomerular disorder, a personalized treatment protocol can be formulatedfor the prevention or treatment of the proteinuric glomerular disorderspecific for the at least one subject. In one embodiment, the at leastone subject is at risk for development of a primary or secondary orrecurrent proteinuric glomerular disorder such as FSGS, Membranousnephropathy, minimal change disease, IgA nephropathy,Membranoproliferative glomerulopathy, diabetic nephropathy, lupusnephritis, myeloma kidney, hypertensive nephrosclerosis, paucimmuneglomerulonephritis, preeclampsia, amyloidosis, cryoglobulinemia,thrombotic thrombocytopenic purpura, Hemolytic uremic syndrome,scleroderma kidney, Alport's glomerulopathy, transplant glomerulopathy,or a storage disorder, and the cytoskeletal disruption or rearrangementincludes one or more of: cellular blebbing, cellular enlargement,apoptosis, and loss of stress fibers.

Also described herein is an assay for determining if at least onesubject who is at risk for a primary or secondary proteinuric glomerulardisorder or who has a primary or secondary proteinuric glomerulardisorder is at risk for primary or secondary disease development,progression or recurrence of the proteinuric glomerular disorder afterkidney transplantation. The assay includes: obtaining a biologicalsample from the subject; contacting the biological sample with reportercells having a vector containing a nucleic acid encoding an SMPDL-3bpromoter sequence operably linked to a nucleic acid encoding a reportergene; analyzing expression levels of the reporter gene in the reportercells; and correlating a change in reporter gene expression relative toa control with an increased risk for primary or secondary diseasedevelopment or progression or an increased risk for a recurrence of theproteinuric or glomerular disorder after kidney transplantation in thesubject. In the assay, the proteinuric glomerular disorder is typicallyone of: FSGS, Membranous nephropathy, minimal change disease, IgAnephropathy, Membranoproliferative glomerulopathy, diabetic nephropathy,lupus nephritis, myeloma kidney, hypertensive nephrosclerosis,paucimmune glomerulonephritis, preeclampsia, amyloidosis,cryoglobulinemia, thrombotic thrombocytopenic purpura, Hemolytic uremicsyndrome, scleroderma kidney, Alport' s glomerulopathy, transplantglomerulopathy, and a storage disorder. The reporter gene can be, forexample, a nucleic acid encoding luciferase and the reporter cells aretransfectable mammalian cells. The assay can further include analyzingnormal human podocytes contacted with the biological sample andquantitatively measuring at least one of: oxygen consumption rate,intracellular lipid accumulation, and apoptosis, and correlating achange in the at least one quantitative measurement relative to acontrol with an increased risk of development, progression or recurrenceof the proteinuric glomerular disorder after kidney transplantation inthe subject. In another embodiment, the assay includes analyzing thephenotype quantitatively based on a standard curve generated with twodose-dependent chemicals that increase or decrease SMPDL-3b expressionor activity. The assay can further include analyzing normal humanpodocytes contacted with the biological sample for the presence orabsence of cytoskeletal disruptions or rearrangements and correlatingthe presence of cytoskeletal disruptions or rearrangements in the normalpodocytes with an increased risk of development, progression, orrecurrence of the proteinuric glomerular disorder after kidneytransplantation in the subject. In one embodiment, the at least onesubject is at risk for the development of a primary or secondaryproteinuric glomerular disorder, and the proteinuric glomerular disorderis one of: FSGS, Membranous nephropathy, minimal change disease, IgAnephropathy, Membranoproliferative glomerulopathy, diabetic nephropathy,lupus nephritis, myeloma kidney, hypertensive nephrosclerosis,paucimmune glomerulonephritis, preeclampsia, amyloidosis,cryoglobulinemia, thrombotic thrombocytopenic purpura, Hemolytic uremicsyndrome, scleroderma kidney, Alport's glomerulopathy, transplantglomerulopathy, and a storage disorder.

Still further described herein is an assay for predicting a response ina subject to at least one candidate therapeutic agent for prevention ortreatment of a particular proteinuric glomerular or renal-relateddisease. The assay includes the steps of: obtaining a biological samplefrom the subject having the particular proteinuric glomerular orrenal-related disease; contacting a first portion of the biologicalsample with a first culture of human podocytes in the presence of the atleast one candidate therapeutic agent and contacting a second portion ofthe biological sample with a second culture of podocytes in the absenceof the at least one candidate therapeutic agent; examining the first andsecond cultures of podocytes for the presence or absence of cytoskeletaldisruptions or rearrangements; and correlating an increase incytoskeletal disruptions or rearrangements in the second culture ofpodocytes relative to the first culture of podocytes with a positiveresponse to the at least one candidate therapeutic agent. The assaytypically includes repeating one or more of these steps until at leastone candidate therapeutic agent that is effective for at least one of:restoration of actin cytoskeleton, preservation of actin cytoskeleton,restoration of physiological SMPDL-3b expression, and preservation ofphysiological SMPDL-3b expression is identified for the treatment orprevention of the particular proteinuric glomerular disease. The atleast one candidate therapeutic agent is at least one of: a drugapproved for treatment of the particular proteinuric glomerular disease,and an off-label drug. Typically, the at least one candidate therapeuticagent is one that modulates activity or expression of at least one of:SMPDL-3b, ASMase, ceramide, S1P, ABCA1, ABCG1, LDL-rec, ACC1, fatty acidsynthase, stearoyil-CoA desaturase, HMG-CoA reductase, and SREBP. Forexample, the at least one candidate therapeutic agent restoresphysiological SMPDL-3b expression or activity, preserves physiologicalSMPDL-3b expression or activity, or prevents degradation of SMPDL-3b.One example of a candidate therapeutic agent is rituximab.

An assay for predicting a response in a subject to at least onecandidate therapeutic agent for treatment of a particular proteinuricglomerular or renal-related disease is also described herein. The methodincludes the steps of: obtaining a biological sample from the subjecthaving the particular proteinuric glomerular or renal-related disease;contacting the biological sample with cells including a vectorcontaining a nucleic acid encoding an SMPDL-3b promoter sequenceoperably linked to a nucleic acid encoding a reporter gene; contactingthe mixture of biological sample and reporter cells with at least onecandidate therapeutic agent; analyzing reporter gene expression in thereporter cells after contacted with the biological sample and the atleast one candidate therapeutic agent; quantifying the reporter geneexpression and comparing the reporter gene expression to a control; andcorrelating a change in the reporter gene expression relative to thecontrol with the subject's response to the at least one candidatetherapeutic agent. The at least one candidate therapeutic agent can beone or more of a drug approved for treatment of the particularproteinuric glomerular disease, and an off-label drug. Typically, the atleast one candidate therapeutic agent is one that restores physiologicalSMPDL-3b expression or activity, preserves physiological SMPDL-3bexpression or activity, or that prevents degradation of SMPDL-3b.

Yet further described herein is an assay for identifying at least onetherapeutic agent for preventing or treating a primary or secondary orrecurrent proteinuric glomerular disorder in at least one subject. Theassay includes a first step of contacting reporter cells with at leastone of: a proteinuric glomerular disease-specific pool of sera from atleast one subject having or at risk for the proteinuric glomerulardisease, sera from a subject who does not have the proteinuricglomerular disease, and a chemical that either induces or reducesSMPDL-3b expression in at least one multi-well plate. In the assay, thereporter cells generally include a vector containing a nucleic acidencoding an SMPDL-3b promoter sequence operably linked to a nucleic acidencoding a reporter. Another step of the assay is contacting a firstportion of the reporter cells with a library of candidate therapeuticagents, wherein at least a second portion of the reporter cells is notcontacted with the library of candidate therapeutic agents. Additionalsteps of the assay are analyzing reporter gene expression in the firstand second portions of reporter cells; identifying at least onetherapeutic agent from the library of candidate therapeutic agents thatprevents modulation of SMPDL-3b expression, restores SMPDL-3b expressionto a control level, or preserves SMPDL-3b expression at a control level;and correlating the therapeutic agent's ability to prevent modulation ofSMPDL-3b expression, restore SMPDL-3b expression to the control level orpreserve SMPDL-3b expression at the control level with an ability toprevent or treat recurrent proteinuric glomerular disease in the atleast one subject. In the assay, the primary or secondary or recurrentproteinuric glomerular disorder is typically one of: FSGS, Membranousnephropathy, minimal change disease, IgA nephropathy,Membranoproliferative glomerulopathy, diabetic nephropathy, lupusnephritis, myeloma kidney, hypertensive nephrosclerosis, paucimmuneglomerulonephritis, preeclampsia, amyloidosis, cryoglobulinemia,thrombotic thrombocytopenic purpura, Hemolytic uremic syndrome,scleroderma kidney, Alport's glomerulopathy, transplant glomerulopathy,and a storage disorder. The assay can further include the step of:correlating the therapeutic agent's ability to prevent modulation ofSMPDL-3b expression, restore SMPDL-3b expression to a control level, orpreserve SMPDL-3b expression at a control level with an ability toprevent or reverse cytoskeletal disruptions or rearrangements observedin at least one culture of podocytes cultured in at least one multi-wellplate with sera from the at least one subject having the proteinuricglomerular disease, or with a chemical that induces cytoskeletalrearrangements. Examples of a chemical that induces cytoskeletalrearrangements or preserves cytoskeleton structure include cytokalasinD, lipopolysaccharide, puromycin aminonucleoside, protamine sulphate,phalloidin, and latrunculin.

An assay for identifying at least one therapeutic agent for preventingor treating a primary or secondary or recurrent proteinuric glomerulardisorder in at least one subject is described herein. The assayincludes: culturing human podocytes in the presence of at least one of:a proteinuric glomerular disease-specific pool of sera from at least onesubject having the proteinuric glomerular disease, sera from a subjectwho does not have the proteinuric glomerular disease, and a chemicalthat induces, prevents or decreases cytoskeletal disruptions orrearrangements in at least one multi-well plate; contacting the culturedpodocytes with one or more candidate therapeutic agents from a libraryof candidate therapeutic agents; examining the cultured podocytescontacted with one or more candidate therapeutic agents from a libraryof candidate therapeutic agents for cyto skeletal disruptions orrearrangments; and identifying at least one therapeutic agent whichprevents or decreases cytoskeletal disruptions or rearrangements.Generally, the primary or secondary or recurrent proteinuric glomerulardisorder is one or more of: FSGS, Membranous nephropathy, minimal changedisease, IgA nephropathy, Membranoproliferative glomerulopathy, diabeticnephropathy, lupus nephritis, myeloma kidney, hypertensivenephrosclerosis, paucimmune glomerulonephritis, preeclampsia,amyloidosis, cryoglobulinemia, thrombotic thrombocytopenic purpura,Hemolytic uremic syndrome, scleroderma kidney, Alport's glomerulopathy,transplant glomerulopathy, and a storage disorder. The assay can furtherinclude examining the podocytes for expression of at least one factorthat affects podocyte function, e.g., SMPDL-3b, ASMase, ceramide, S1P,ABCA1, ABCG1, LDL-rec, ACC1, fatty acid synthase, HMG-CoA reductase, andan SERBP.

Yet further described herein is a method of preventing or treatingprimary or secondary proteinuric glomerular disorder or recurrentproteinuric glomerular disease in a subject. The method includes thesteps of: providing a composition including an agent that restoresphysiological lipid content in podocytes or physiological lipid-relatedprotein content in podocytes, and a pharmaceutically acceptable carrier;and administering the composition to the subject in a therapeuticallyeffective amount for preventing apoptosis of podocytes, preventingdisruption of podocyte cytoskeleton, preventing accumulation ofcholesterol and/or lipids in podocytes, and preventing or treatingprimary or secondary proteinuric glomerular disorder or recurrentproteinuric or glomerular disease in the subject. The primary orsecondary proteinuric glomerular disorder or recurrent proteinuricglomerular disease can be, e.g., diabetic nephropathy. The agent thatrestores physiological lipid content in podocytes or physiologicallipid-related protein content in podocytes can be one or more of, forexample, rituximab, a cyclodextrin derivative, and an agent thatmodulates SMPDL-3b expression or activity. In the method, thecomposition can be administered to the subject at one or more of thefollowing time points: prior to kidney transplantation, during kidneytransplantation, and subsequent to kidney transplantation.

A method of preventing progression of FSGS or recurrence of FSGS afterkidney transplantation, or treating FSGS in a subject having FSGS isdescribed herein. The method includes administering to the subject acomposition including a pharmaceutically acceptable carrier and an agentthat is capable of at least one of: increasing SMPDL-3b levels in thesubject, restoring cytoskeleton rearrangements in the subject, anddecreasing or preventing B7-1 expression or activity in the subject. Thecomposition is administered to the subject having FSGS in an amounteffective to prevent or treat FSGS in the subject. The agent can be, forexample, rituximab or abatacept. In the method, the composition can beadministered to the subject at one or more of the following time points:prior to kidney transplantation, during kidney transplantation, andsubsequent to kidney transplantation.

A kit for determining if at least one subject who is at risk for aprimary or secondary proteinuric glomerular disorder or who has aprimary or secondary proteinuric glomerular disorder is at risk forprimary or secondary disease development, progression or recurrence ofthe proteinuric glomerular disorder after kidney transplantation isdescribed herein. The kit includes: a plurality of reporter cellsincluding a vector containing a nucleic acid encoding an SMPDL-3bpromoter sequence operably linked to a nucleic acid encoding a reportergene; at least one control; and instructions for use.

Also described herein is a kit for predicting a response in at least onesubject to at least one candidate therapeutic agent for treatment orprevention of a particular proteinuric glomerular disease. The kitincludes: a plurality of reporter cells including a vector containing anucleic acid encoding an SMPDL-3b promoter sequence operably linked to anucleic acid encoding a reporter gene; a plurality of candidatetherapeutic agents; at least one control; and instructions for use.

Additionally described herein is a kit for determining if at least onesubject who is at risk for a primary or secondary proteinuric glomerulardisorder or who has a primary or secondary proteinuric glomerulardisorder is at risk for primary or secondary disease development,progression or recurrence of the proteinuric glomerular disorder afterkidney transplantation. The kit includes: at least one container ofpodocytes; cytochalasin D as a positive control; a dye for actincytoskeleton; and instructions for use.

Still further described herein is a kit for predicting a response in atleast one subject to at least one candidate therapeutic agent fortreatment of a particular proteinuric or glomerular disease. The kitincludes: at least one container of podocytes; cytochalasin D as apositive control; a dye for actin cytoskeleton; a plurality of candidatetherapeutic agents; and instructions for use.

A method of preventing or treating a renal-related disorder in a subjectis described herein. The method includes administering to the subject acomposition including one or more of: a cyclodextrin, a cyclodextrinderivative, and a cellular cholesterol-lowering agent that is not astatin, in an amount effective for reducing at least one of: plasmamembrane cholesterol, plasma membrane lipids, cellular cholesterol, andcellular lipids in the subject. The renal-related disease may be primaryglomerulopathy, secondary glomerulopathy, or a post-transplantrecurrence of a glomerular disease such as: FSGS, Membranousnephropathy, minimal change disease, IgA nephropathy,Membranoproliferative glomerulopathy, diabetic nephropathy, lupusnephritis, myeloma kidney, hypertensive nephrosclerosis, paucimmuneglomerulonephritis, preeclampsia, amyloidosis, cryoglobulinemia,thrombotic thrombocytopenic purpura, Hemolytic uremic syndrome,scleroderma kidney, Alport's glomerulopathy, transplant glomerulopathystorage disorder, stroke, peripheral vascular disease, diabetes,coronary artery disease, congestive heart failure, atherosclerosis,cardiac hypertrophy, myocardial infarction, endothelial dysfunction andhypertension. The composition is administered in an amount effective forpreserving podocyte function and preventing or treating proteinuria inthe subject. Typically, administration of the composition results inreduction of at least one of: plasma membrane cholesterol, plasmamembrane lipids, cellular cholesterol, and cellular lipids in podocytesof the kidney of the subject. The composition can further include one ormore of the following drugs: immunosuppressive agent, ACTH agonist,insulin sensitizer, GH antagonist, antinflammatory medication, vitamin Dderivative, RAS system inhibitor, aldosterone inhibitor, HMG-CoAreductase inhibitor, cholesterol absorption inhibitor, bile acidsequestrant, bile acid resin, Niacin, Niacin derivative, fibrate,cholesteryl ester transfer protein (CETP) inhibitor, Acetyl-Coenzyme Aacetyltransferase (ACAT) inhibitor, and microsomal triglyceridetransport inhibitor.

Unless otherwise defined, all technical terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs.

As used herein, “protein” and “polypeptide” are used synonymously tomean any peptide-linked chain of amino acids, regardless of length orpost-translational modification, e.g., glycosylation or phosphorylation.

By the term “gene” is meant a nucleic acid molecule that codes for aparticular protein, or in certain cases, a functional or structural RNAmolecule.

As used herein, a “nucleic acid” or a “nucleic acid molecule” means achain of two or more nucleotides such as RNA (ribonucleic acid) and DNA(deoxyribonucleic acid).

As used herein, the terms “kidney disease(s),” “renal disease,” “renaldisorder,” and “kidney disorder(s)” “kidney injury”, “renal injury”,“proteinuria”, “albuminuria” “podocytopathy,” “glomerulopathy”,“tubulopathy”, “tubular disorder”, “nephritic syndrome” “nephroticsyndrome” and “nephropathy” are interchangeable and mean any disease,disorder, syndrome, anomaly, pathology, or abnormal condition of thekidney or of the structure or function of its constituent parts.

As used herein, “proteinuria” refers to an amount of protein larger than150 mg passing through the kidney filtration barrier in a 24 hourperiod. “Albuminuria” can be defined as the selective passage of albuminthrough the filtration barrier, and it is defined as “microalbuminuria”when ranging between 30 and 300 mg/g creatinine and “macroalbuminuria”when above 300 mg/g creatinine in a spot urine collection. Albuminuriaand proteinuria are often a consequence of damage to the podocyte, a keyconstituent of the glomerular filtration barrier that normally would notallow for any protein passage. Proteinuria is a characteristic findingin many renal (kidney) diseases and/or glomerulopathy that can either beidiopatic, genetic or secondary to systemic diseases such ashypertension, eclampsia, diabetes mellitus, lupus, vasculitidis,hemathologic disorder, amyloidosis, cancer, allergic reactions, toxicinsult by many drug/agents and transplant glomerulopathy among manyother less common causes. Many of these diseases can manifest withnephrotic range proteinuria (i.e., proteinuria larger than 3.5 grams perday or larger than 3.5 g/g creatinine) and manifest as nephroticsyndrome when associated with hypoalbuminemia, hyercoagulability,hypercholesterolemia and edema.

By the term “proteinuric glomerular disorder” is any primary, secondaryor post-transplant glomerular disorder associated with the loss ofalbumin and/or protein as defined above in 0018.

The terms “patient,” “subject” and “individual” are used interchangeablyherein, and mean an animal (e.g., a mammal such as a human, avertebrate) subject to be treated and/or to obtain a biological samplefrom.

As used herein, “bind,” “binds,” or “interacts with” means that onemolecule recognizes and adheres to a particular second molecule in asample or organism, but does not substantially recognize or adhere toother structurally unrelated molecules in the sample. Generally, a firstmolecule that “specifically binds” a second molecule has a bindingaffinity greater than about 10⁸ to 10¹² moles/liter for that secondmolecule and involves precise “hand-in-a-glove” docking interactionsthat can be covalent and noncovalent (hydrogen bonding, hydrophobic,ionic, and van der waals).

The term “labeled,” with regard to a nucleic acid, protein, probe orantibody, is intended to encompass direct labeling of the nucleic acid,protein, probe or antibody by coupling (i.e., physically or chemicallylinking) a detectable substance (detectable agent) to the nucleic acid,protein, probe or antibody.

When referring to a nucleic acid molecule or polypeptide, the term“native” refers to a naturally-occurring (e.g., a wild type, WT) nucleicacid or polypeptide.

By the term “off label” when referring to a drug or compound means thatthe drug or compound is used in a different way than described in theFDA-approved drug or compound label.

As used herein, the terms “therapeutic,” and “therapeutic agent” areused interchangeably, and are meant to encompass any molecule, chemicalentity, composition, drug, therapeutic agent, chemotherapeutic agent, orbiological agent capable of preventing, ameliorating, or treating adisease or other medical condition. The term includes small moleculecompounds, antisense reagents, siRNA reagents, antibodies, enzymes,peptides organic or inorganic molecules, cells, natural or syntheticcompounds and the like.

As used herein, the terms “diagnostic,” “diagnose” and “diagnosed” meanidentifying the presence or nature of a pathologic condition.

The term “sample” is used herein in its broadest sense. A sampleincluding polynucleotides, peptides, antibodies and the like may includea bodily fluid, a soluble fraction of a cell preparation or media inwhich cells were grown, genomic DNA, RNA or cDNA, a cell, a tissue,skin, hair and the like. Examples of samples include saliva, serum,tissue, skin, blood, urine and plasma.

As used herein, the term “treatment” is defined as the application oradministration of a therapeutic agent to a patient or subject, orapplication or administration of the therapeutic agent to an isolatedtissue or cell line from a patient or subject, who has a disease, asymptom of disease or a predisposition toward a disease, with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve, prevent or affect the disease, the symptoms of disease, or thepredisposition toward disease.

Although assays, compositions, kits, and methods similar or equivalentto those described herein can be used in the practice or testing of thepresent invention, suitable assays, compositions, kits, and methods aredescribed below. All publications, patent applications, and patentsmentioned herein are incorporated by reference in their entirety. In thecase of conflict, the present specification, including definitions, willcontrol. The particular embodiments discussed below are illustrativeonly and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-FIG. 1E: Shows that both rituximab and SMPDL-3b partiallyprevent the effect of recurrent FSGS sera on podocyte stress fibers.FIG. 1A: Shows representative stress fiber confocal images of normalhuman podocytes exposed to normal (NHS) (n=5), non-recurrent (NON-REC)FSGS (n=10), and recurrent (REC) FSGS (n=12) human sera. Scale bars, 10μm. FIG. 1B: Depicts the percentage of cells with disruption of stressfibers observed after exposure to NHS (n=5), non-recurrent sera (n=10),and recurrent human sera (n=12). ***p<0.001 by one-way ANOVA. FIG. 1C:Depicts the linear correlation between the percentage of cells with lossof stress fibers and the urine protein/creatinine ratio obtained fromREC and NON-REC patients (n=22) in the first 30 days aftertransplantation (R²=0.59; p<0.001 calculated with an F test with 18degrees of freedom). FIG. 1D: Depicts confocal images of stress fibersand corresponding bar graph analysis of normal human podocytes exposedto recurrent FSGS sera in the presence (REC+RITUX) or absence (REC) ofrituximab. Rituximab protected the loss of stress fibers observed instressed podocytes exposed to recurrent FSGS, but not non-recurrent FSGShuman sera. Scale bars, 10 μm, data are mean±s.d. *p<0.05; ***p<0.001 byone-way ANOVA. FIG. 1E: Depicts confocal images of stress fibers andcorresponding bar graph analysis of normal human podocytes exposed toREC sera transfected with an empty GFP vector (REC) or with aSMPDL-3b-GFP vector (REC+SMPDL-3b). SMPDL-3b overexpression protectedthe loss of stress fibers observed in podocytes exposed to recurrentFSGS human sera, data are mean±s.d. *p<0.05; ***p<0.001 by one-wayANOVA. The in vitro studies strongly correlate to in vivo clinicaloutcome data, as rituximab administered in patients at high risk forrecurrent FSGS after transplantation significantly protected from thedevelopment of post transplant proteinuria as demonstrated in Table 1.

FIG. 2A-FIG. 2G: Shows that rituximab prevents the downregulation ofSMPDL-3b in recurrent FSGS. FIG. 2A: Depicts low- and high-power imagesof immunoperoxidase staining for SMPDL-3b and synaptopodin inpost-reperfusion biopsies of patients with recurrent (REC) andnon-recurrent (NON-REC) FSGS. Arrows point to podocytes. Scale bars: 25μm top and 15 μm bottom. FIG. 2B: Depicts the number of SMPDL-3b+podocytes per glomerulus, as evaluated by SMPDL-3b and synaptopodinlabeling in post-reperfusion kidney biopsies from patients that later ondeveloped recurrent (REC) disease (n=8) and patients that did notdevelop clinical recurrence (NON-REC) (n=12). All kidney biopsies wereobtained prior to initiation of treatment with rituximab. An average of13±4 glomeruli per patient were analyzed. *p<0.05, unpaired Student's ttest. Data are mean±s.d. FIG. 2C: Shows regulation of podocyte SMPDL-3bmRNA expression by normal (NHS), non-recurrent (NON-REC) FSGS, andrecurrent (REC) FSGS human sera (n=4 per group) and by rituximab. Dataare mean±s.d. *p<0.05 and **p<0.01 by one-way ANOVA. FIG. 2D: Depictsthe amount of SMPDL-3b protein is normalized to actin in human podocytestreated with normal (n=5), recurrent (n=12), or non-recurrent (n=10)human sera and exposed to rituximab. Data are mean±s.d. *p<0.05 and**p<0.01 by one-way ANOVA. FIG. 2E: Shows western blot for SMPDL-3bprotein of normal podocytes cultured with sera from consecutivenon-recurrent (n=4) and recurrent (n=4) FSGS patients in the presence orabsence of rituximab. FIG. 2F: Depicts the amount of 52 and 54 kDaASMase protein is normalized to actin in human podocytes that wereexposed to normal (n=5), non-recurrent FSGS (n=10), and recurrent FSGS(n=12) human sera in the presence or absence of rituximab. Data aremean±s.d. *p<0.05 and **p<0.01 by one-way ANOVA. FIG. 2G: Shows ASMaseactivity per μg of total lysate protein, as evaluated by ELISA. Data aremean±s.d. *p<0.05 and **p<0.01 by one-way ANOVA.

FIG. 3A-FIG. 3F: Shows regulation of actin stress fibers and cholesterolcontent in human podocytes exposed to the sera of patients with DM withor without DN. FIG. 3A: Shows confocal stress fiber images and brightfield images of podocytes exposed to the three different groups of serademonstrating cell blebbing. FIG. 3B: Shows quantification of cells withblebs in cells exposed to DM w DN sera when compared to cells exposed DMw/o DN sera or NHS. FIG. 3C: Depicts representative immunofluorescenceimages for cholesterol content (filipin staining), and phosphorylatedcaveolin and DAPI in podocytes exposed to the three different groups ofsera demonstrating that DM w DN sera causes lipid accumulation andperipheral distribution of p-caveolin, a phenomenon that can beprevented by CD. FIG. 3D: Shows quantitative bar graph analysis of totalcholesterol in podocytes exposed to DM w DN sera when compared to DM w/oDN and NHS. Total cholesterol (TC) significantly increased in podocytesexposed to DM w DN sera, a phenomenon that could be prevented by CD.FIG. 3E: Shows confocal images of cell blebbing observed after exposureto the sera of diabetic patients before and after progressing to DN,demonstrating that cell blebbing precedes DN. FIG. 3F: Depictsquantitative bar graph analysis of cleaved caspase 3 in podocytesexposed to DM w DN sera when compared to DM w/o DN and NHS showingincreased apoptosis in podocytes exposed to DM w DN sera, a phenomenonthat could be prevented by CD.

FIG. 4A-FIG. 4C: Shows treatment of proteinuria with Abatacept inpatients with recurrent FSGS and biopsy-proven B7-1 podocyte expression.FIG. 4A: Shows double labeling confocal microscopy with the podocyteprotein synaptopodin shows podocyte B7-1 expression in post- but notpre-transplant biopsy in recurrent FSGS. Podocyte B7-1 expression wasseen in 3 out of 5 glomeruli in the depicted post-transplant FSGS case.FIG. 4B: Shows detection of podocyte FP effacement in the post- but notpre-transplant biopsy. FIG. 4C: Depicts serum levels of creatinine(filled circle) and urinary protein/creatinine ratios in post-transplant(empty triangle) after Abatacept and plasmapheresis (PP) treatment.Concomitant serum albumin levels are given in mg/dl (filled square).

FIG. 5A-FIG. 5B: Shows that SMPDL-3b deficiency is associated withincreased cellular cholesterol and decreased ASMase activity. FIG. 5A:Shows human podocytes deficient of SMPDL-3b (siSMP) are characterized byincreased cholesterol content when compared to control podocytes (CTRL).FIG. 5B: Shows that siSMP podocytes are characterized by decreasedASMase activity when compared to CTRL (***p<0.001).

FIG. 6A-FIG. 6B: Depicts that CD partially prevents LPS induced podocytedamage. FIG. 6A: Depicts the results when HPBCD was administeredintravenously (4000 mg/kg) 1 hour prior to LPS injection (200 ug/20 gmouse intraperitoneally). Urines were collected 48 hours after LPSinjection and analyzed for albumin/creatinine ratio by ELISA. p<0.05when comparing LPS+CD versus LPS. FIG. 6B: Depicts that CD prevented LPSinduction of MyD88 in isolated glomeruli in vivo.

DETAILED DESCRIPTION

Described herein are assays, methods and kits for predicting a subject's(e.g., human) risk of development or progression of a glomerular disease(e.g., FSGS, DN, Membranous nephropathy, minimal change disease, IgAnephropathy, Membranoproliferative glomerulopathy, lupus nephritis,myeloma kidney, hypertensive nephrosclerosis, paucimmuneglomerulonephritis, preeclampsia, amyloidosis, cryoglobulinemia,thrombotic thrombocytopenic purpura, Hemolytic uremic syndrome,scleroderma kidney, Alport's glomerulopathy, storage disorders and otherrate genetic disorders). Described herein are assays for predicting, forexample, if a diabetic subject will develop glomerular or kidneydisease, assays for identifying a new or previously availabletherapeutic agent for preventing or treating a glomerular disease, andassays for establishing a personalized approach to treat or prevent aglomerular disease in a subject. A high-throughput platform will findparticular use for the development of therapeutics to treat renaldiseases such as FSGS. Based on the experimental results describedbelow, serum-induced cytoskeletal disruptions and rearrangements, aswell as modulation of SMPDL-3b expression, stability or activity, aswell as lipid content, oxygen consumption rate, and apoptosis in normal,healthy human podocytes may be used as a marker for predicting recurrentglomerular disease (e.g., recurrent FSGS) in a pre-transplant patient.The assays, methods and kits may be used for any glomerular disease(e.g., FSGS, DN, Membranous nephropathy, minimal change disease, IgAnephropathy, Membranoproliferative glomerulopathy, lupus nephritis,myeloma kidney, hypertensive nephro sclerosis, paucimmuneglomerulonephritis, preeclampsia, amyloidosis, cryoglobulinemia,thrombotic thrombocytopenic purpura, Hemolytic uremic syndrome,scleroderma kidney, Alport's glomerulopathy, storage disorders and otherrate genetic disorders), providing the ability to predict the clinicalcourse of a disease and to develop personalized treatment strategies.This new technology may be used as an easily performed assay for useworldwide as a prognostic tool for primary, secondary or transplantrelated glomerular diseases and as a predictive tool for theidentification of disease-specific prevention and treatment strategies.Further described herein are compositions, kits and methods for treatingglomerular and kidney diseases.

Biological and Chemical Methods

Methods involving conventional molecular biology techniques aredescribed herein. Such techniques are generally known in the art and aredescribed in detail in methodology treatises such as Molecular Cloning:A Laboratory Manual, 3rd ed., vol. 1-3, ed. Sambrook et al., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; and CurrentProtocols in Molecular Biology, ed. Ausubel et al., Greene Publishingand Wiley-Interscience, New York, 1992 (with periodic updates).Conventional methods of culturing mammalian cells, e.g., humanpodocytes, are generally known in the art. Methods of culturingpodocytes are described in detail in Saleem M A, O'Hare M J, Reiser J,et al., J Am Soc Nephrol 2002; 13(3):630-8. Examples of methods for thesynthesis of molecular libraries can be found in the art, for examplein: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb etal. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al.(1994). J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303;Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al.(1994) Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J.Med. Chem. 37:1233. The amino acid sequence of SMPDL-3b and the nucleicacid sequence encoding SMPDL-3b are known as accession numbersNM_001009568.1 and NM_014474.2. For a reference describing thecharacterization of SMPDL-3b, see, for example, Perosa et al., Bloodvol. 107:1070-1077, 2006.

Assays for Predicting Responses to Treatment in a Subject

Described herein are assays for predicting a response in a subject to atleast one candidate therapeutic agent for treatment of a particularproteinuric glomerular or renal-related disease. In one embodiment, theassay includes: obtaining a biological sample from the subject at riskfor or having the particular proteinuric glomerular or renal-relateddisease; contacting a first portion of the biological sample with afirst culture of human podocytes in the presence of the at least onecandidate therapeutic agent and contacting a second portion of thebiological sample with a second culture of podocytes in the absence ofthe at least one candidate therapeutic agent; examining the first andsecond cultures of podocytes for the presence or absence of cytoskeletaldisruptions or rearrangements; and correlating an increase incytoskeletal disruptions or rearrangements in the second culture ofpodocytes relative to the first culture of podocytes with a positiveresponse to the at least one candidate therapeutic agent. In this assay,one or more of these steps can be repeated until at least one candidatetherapeutic agent that is effective for restoration of actincytoskeleton and/or physiological SMPDL-3b expression and for thetreatment of the particular proteinuric glomerular disease isidentified.

In another embodiment, the assay includes: obtaining a biological samplefrom the subject having the particular proteinuric glomerular orrenal-related disease; contacting the biological sample with cellscomprising a vector containing a nucleic acid encoding the SMPDL-3bpromoter sequence operably linked to a nucleic acid encoding a reportergene; contacting the mixture of biological sample and reporter cellswith at least one candidate therapeutic agent; analyzing reporter geneexpression in the reporter cells after contacted with the biologicalsample and the at least one candidate therapeutic agent; quantifying thereporter gene expression and comparing the reporter gene expression to acontrol; and correlating a change in the reporter gene expressionrelative to the control with the subject's response to the at least onecandidate therapeutic agent.

In these embodiments, the at least one candidate therapeutic agent canbe one or more of a drug approved for treatment of the particularproteinuric glomerular disease, and/or an off-label drug. In a typicalembodiment, the at least one candidate therapeutic agent is one thatrestores physiological SMPDL-3b expression or activity, or that preventsdegradation of SMPDL-3b (e.g., rituximab). Additionally oralternatively, the at least one candidate therapeutic agent can be onethat modulates activity or expression of, for example, ASMase, ceramide,S1P, ABCA1, ABCG1, LDL-rec, ACC1, fatty acid synthase, stearoyil-CoAdesaturase, HMG-CoA reductase, and SREBP.

In one embodiment, the assay includes examining the podocytes for thepresence or absence of cytoskeletal disruptions or rearrangements, lipidaccumulation, modulation of SMPDL-3b, and/or apoptosis, resulting indetermination of a first phenotype of the podocytes. This firstphenotype can be compared with one or more controls. For example, thefirst phenotype can be compared with a second phenotype of podocytescontacted with a biological sample from at least one normal subject, anda third phenotype of podocytes contacted with a biological sample fromat least one subject with the glomerular disorder (e.g., recurrentFSGS). In such an embodiment, the second phenotype is typically anegative control, and the third phenotype a positive control. Such anassay can be used to determine a patient-specific response to one ormore therapeutic strategies that have been approved for the treatment ofthe medical condition being treated in the patient, as well as therapiesthat may be utilized off label. As an example, for any given patientwith diabetes, the in vitro response to established treatment strategies(such as ACE inhibitors, Angiotensin receptor blockers, aldosteroneantagonists, vitamin D analogues, etc.) will be tested in conjunctionwith off label treatment strategies (e.g., cyclodextrin derivatives,abatacept, rituximab, belimumab, atacicept, alefacept, etc.). Using suchan assay, off label treatment strategies that are effective in vitro maybe tested in vivo, and may therefore allow for the identification ofoptimal personalized treatment strategies for any patient with any renaldisorder.

Assay for Determining Risk for Primary or Secondary ProteinuricGlomerular Disorder Development, Progression or Recurrence after KidneyTransplantation

Assays for determining if at least one subject (e.g., 1, 2, 3, 4, 5, 10,50, 100, etc.) who is at risk for or has a primary or secondaryproteinuric glomerular disorder is at risk for primary or secondarydisorder progression or is at risk for a recurrence of the proteinuricglomerular disorder after kidney transplantation. In one embodiment, theassay includes contacting a biological sample from the at least onesubject with a culture of human podocytes; examining the podocytes forthe presence or absence of cytoskeletal disruptions or rearrangementsresulting in determination of a first phenotype of the podocytes; andcorrelating the presence of cytoskeletal disruptions or rearrangementswith an increased risk of development, recurrence, or progression of theproteinuric glomerular disorder (e.g., FSGS, DN, Membranous nephropathy,minimal change disease, IgA nephropathy, Membranoproliferativeglomerulopathy, lupus nephritis, myeloma kidney, hypertensivenephrosclerosis, paucimmune glomerulonephritis, preeclampsia,amyloidosis, cryoglobulinemia, thrombotic thrombocytopenic purpura,Hemolytic uremic syndrome, scleroderma kidney, Alport's glomerulopathy,storage disorders and other rate genetic disorders) in the subject. Anybiological sample can be used, e.g., blood, saliva, serum, plasma,tissue, and urine. The assay may be used for one subject, or a pluralityof subjects who have a primary, secondary or post-transplant proteinuricglomerular disorder. The cytoskeletal disruptions or rearrangements canbe examined by any suitable method, e.g., microscopy, Western blotting,ELISA, flow cytometry, reporter gene assays. Generally, the firstphenotype is compared to a negative control and a positive control. Theassay can further include analyzing the first phenotype quantitativelybased on a standard curve generated with a dose-dependent chemicaldisruption of actin cytoskeleton.

In another embodiment of an assay for determining if at least onesubject (e.g., 1, 2, 3, 4, 5, 10, 50, 100, etc.) who has or is at riskfor a primary or secondary proteinuric glomerular disorder is at riskfor primary or secondary disease progression or is at risk for arecurrence of the proteinuric glomerular disorder after kidneytransplantation, the assay includes the steps of: obtaining a biologicalsample from the subject; contacting the biological sample with reportercells (e.g., transfectable mammalian cells) including a vectorcontaining a nucleic acid encoding the SMPDL-3b promoter sequenceoperably linked to a nucleic acid encoding a reporter gene (e.g., anucleic acid encoding luciferase); analyzing expression levels of thereporter gene in the reporter cells; and correlating a change inreporter gene expression relative to a control with an increased riskfor primary or secondary disease development and/or progression or anincreased risk for a recurrence of the proteinuric or glomerulardisorder. In this embodiment, the assay can further include analyzingnormal human podocytes contacted with the biological sample andquantitatively measuring at least one of: oxygen consumption rate,intracellular lipid accumulation, and apoptosis, and correlating achange in the at least one quantitative measurement relative to acontrol with an increased risk of development, progression or recurrenceof the primary or secondary proteinuric glomerular disorder in thesubject. The first phenotype can be quantitatively based on a standardcurve generated with two dose-dependent chemicals that increase ordecrease SMPDL-3b expression or activity. The assay can further includethe step of analyzing normal human podocytes contacted with thebiological sample for the presence or absence of cytoskeletaldisruptions or rearrangements and correlating the presence ofcytoskeletal disruptions or rearrangements with an increased risk ofdevelopment, recurrence, or progression of the proteinuric glomerulardisorder in the subject.

In some embodiments, the at least one subject is identified as having anincreased risk for development of primary, secondary or recurrentproteinuric glomerular disease, and a personalized treatment protocol isformulated for the prevention or treatment of the proteinuric glomerulardisorder specific for the at least one subject. The at least one subjectmay be at risk for development of a primary or secondary proteinuricglomerular disorder such as, for example, FSGS, Membranous nephropathy,minimal change disease, IgA nephropathy, Membranoproliferativeglomerulopathy, diabetic nephropathy, lupus nephritis, myeloma kidney,hypertensive nephrosclerosis, paucimmune glomerulonephritis,preeclampsia, amyloidosis, cryoglobulinemia, thrombotic thrombocytopenicpurpura, Hemolytic uremic syndrome, scleroderma kidney, Alport'sglomerulopathy, transplant glomerulopathy, and a storage disorder, andthe cytoskeletal disruption or rearrangement includes at least one of:cellular blebbing, cellular enlargement, apoptosis, and loss of stressfibers.

The assays and methods described herein can be used for determining if asubject (e.g., human) who is considering receiving or is planning toreceive a kidney transplant is at risk for recurrent FSGS after kidneytransplantation. One example of such an assay includes the followingsteps: contacting a biological sample from at least one subject who hasFSGS with a culture of normal human podocytes; examining the podocytesfor the presence or absence of cytoskeletal disruptions orrearrangements resulting in determination of a first phenotype of thepodocytes; and correlating the presence of cytoskeletal disruptions orrearrangements with an increased risk of FSGS recurring in at least onesubject subsequent to a kidney transplantation compared to subjectswithout recurrent FSGS or to subjects being transplanted for FSGSunrelated causes. Actin cytoskeleton remodeling can be utilized alone orin combination with the modulation of SMPDL-3b expression, lipidcontent, oxygen consumption rate, apoptosis. The biological sample isobtained prior to the kidney transplantation and initiation ofimmunosuppression in the subject so that the subject's risk of havingrecurrent FSGS post-transplant can be assessed. In the experimentsdescribed herein, sera from subjects were used. In some embodiments,biological samples from a plurality of subjects suspected of having orbeing at risk of developing FSGS can be analyzed simultaneously, e.g.,in a high-throughput format. Any suitable biological sample can becontacted with the cultured podocytes. Examples of biological samplesinclude blood, saliva, serum, plasma, tissue, and urine.

Typically, the subject is suspected of developing or is at high risk ofdeveloping FSGS after receiving a kidney transplant when certain riskfactors are present, primarily age of the patient (less than 15 years ofage) and time from diagnosis to ESRD (less than 3 years). The assayincludes appropriate positive and negative controls. For example, theassay can include comparing the first phenotype with a second phenotypeof podocytes contacted with a biological sample from at least one normalsubject as a negative control, and a third phenotype of podocytescontacted with a biological sample from at least one subject withrecurrent FSGS as a positive control. Because of the limitedavailability of biological samples to be utilized as standardizedpositive controls, a stable chemical agent can be utilized at differentconcentrations to disrupt the actin cytoskeleton (Cytochalasin D). Suchan approach will allow for the development of a dose-dependent standardcurve that will facilitate the standardized quantification of stressfiber disruption when human podocytes will be exposed to patient sera

In some embodiments, at least one subject includes a plurality (e.g., 2,3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 50, 100, etc.) of subjects who haveFSGS. In the experiments described below, disruption of podocyte stressfibers was examined. However, other podocyte abnormalities ordisturbances in cultured podocyte functions can be examined in an assayor method, such as the disruption of podocyte specific proteins involvedin actin cytoskeleton remodeling (synaptopodin) or the translocation ofrelevant plasma membrane proteins (nephrin, podocin, CD2AP) from theplasma membrane (physiologic) to the cytosol (pathologic), theaccumulation of podocyte cellular lipids (e.g. sterols, sphingolipids,triglycerides, free fatty acids, glycosphingolipids, ceramides) and/orthe expression of any factor capable of altering the expression ofSMPDL-3b or any other lipid related protein (e.g. ASMase, S1P, ABCA1,ABCG1, LDL-rec, ACC1, fatty acid synthase, stearoyil-CoA desaturase,HMG-CoA reductase, SERBPs) in podocytes.

One or more methods can be used in the detection of podocyteabnormalities. In one aspect, podocyte abnormalities can be detectedusing, for example, microscopy (e.g., electron microscopy, lightmicroscopy, fluorescence microscopy). Using microscopy or other suitablemeans for examining podocytes, the step of examining the podocytes forthe presence or absence of cytoskeletal disruptions or rearrangementsresults in determination of a first phenotype of the podocytes andincludes counting the number of podocytes that display cytoskeletaldisruptions or rearrangements. Generally, the assay includes analyzing astandard curve built with different doses of a stable chemical orbiological positive control.

Any non-primary culture of normal human podocytes can be used in theassays, methods and kits described herein. The podocytes can be culturedunder any appropriate culture conditions. In the experiments describedherein, the normal human podocytes were cultured according to Saleem MA, O'Hare M J, Reiser J, et al., J Am Soc Nephrol 2002; 13(3):630-8.This immortalized human podocyte cell line allows for the generation ofa sufficient number of podocytes when cultured in permissive conditionsat 33° C. Cells will then achieve terminal differentiation and willgrowth arrest with development of the classical octopus morphology oncethermoshifted at 37° C. for 14 days.

As described above for an assay for predicting a response to treatmentin a subject having a glomerular disorder, an assay for determining if asubject (e.g., human) who is at risk for occurrence or recurrence of aglomerular or proteinuric disorder (e.g., FSGS, DN, Membranousnephropathy, minimal change disease, IgA nephropathy,Membranoproliferative glomerulopathy, lupus nephritis, myeloma kidney,hypertensive nephrosclerosis, paucimmune glomerulonephritis,preeclampsia, amyloidosis, cryoglobulinemia, thrombotic thrombocytopenicpurpura, Hemolytic uremic syndrome, scleroderma kidney, Alport'sglomerulopathy, storage disorders and other rate genetic disorders) caninclude examining modulation of SMPDL-3b in cells (e.g., podocytes). Anexample of such an assay includes: contacting a biological sample fromthe at least one subject with a culture of human podocytes; examiningthe podocytes for modulation of SMPDL-3b resulting in determination of afirst phenotype of the podocytes; and correlating the modulation ofSMPDL-3b with an increased risk of occurrence or recurrence of aglomerular or proteinuric disorder (e.g., FSGS, DN, Membranousnephropathy, minimal change disease, IgA nephropathy,Membranoproliferative glomerulopathy, lupus nephritis, myeloma kidney,hypertensive nephrosclerosis, paucimmune glomerulonephritis,preeclampsia, amyloidosis, cryoglobulinemia, thrombotic thrombocytopenicpurpura, Hemolytic uremic syndrome, scleroderma kidney, Alport'sglomerulopathy, storage disorders and other rate genetic disorders) Theassay can further include comparing the first phenotype with a secondphenotype of podocytes contacted with a biological sample from at leastone normal subject, and a third phenotype of podocytes contacted with abiological sample from at least one subject with the proteinuric orglomerular disorder (e.g., FSGS, DN, Membranous nephropathy, minimalchange disease, IgA nephropathy, Membranoproliferative glomerulopathy,lupus nephritis, myeloma kidney, hypertensive nephrosclerosis,paucimmune glomerulonephritis, preeclampsia, amyloidosis,cryoglobulinemia, thrombotic thrombocytopenic purpura, Hemolytic uremicsyndrome, scleroderma kidney, Alport's glomerulopathy, storage disordersand other rate genetic disorders). The second phenotype is typically anegative control, and the third phenotype a positive control. In theassay, modulation of SMPDL-3b can be analyzed by Western blotting, PCRor any SMPDL-3b reporter activity. For the latter, any assay kit inwhich the SMPDL3b promoter has been cloned into a vector that contains afirefly luciferase gene and a eukaryotic selection marker cassette butthat lacks eukaryotic promoter and enhancer sequences is suitable. Thus,luciferase activity observed in cells transfected with such constructwill correlate with the expression of the SMPDL3b gene in any culturesystem. The SMPDL3b-promoter-Luciferase construct could be used togenerate stable transfected human podocytes, HEK cell lines or othermammalian cell lines. Cell lines will be used to quantify SMPDL3bpromoter-activity and SMPDL3b expression after treatment with the serumof patients being screened.

This assay can further include comparing the first phenotype with asecond phenotype of podocytes contacted with a biological sample from atleast one normal subject, and a third phenotype of podocytes contactedwith a biological sample from at least one subject with the proteinuricor glomerular disorder (e.g., FSGS, DN, Membranous nephropathy, minimalchange disease, IgA nephropathy, Membranoproliferative glomerulopathy,lupus nephritis, myeloma kidney, hypertensive nephrosclerosis,paucimmune glomerulonephritis, preeclampsia, amyloidosis,cryoglobulinemia, thrombotic thrombocytopenic purpura, Hemolytic uremicsyndrome, scleroderma kidney, Alport's glomerulopathy, storage disordersand other rate genetic disorders). The second phenotype is typically anegative control, and the third phenotype a positive control. Such anassay can be used to predict clinical development of disease and todetermine a patient-specific response to one or more therapeuticstrategies that have been approved for the treatment of the medicalcondition being treated in the patient, as well as therapies that may beutilized off label.

Screening Platform for Therapeutics for Proteinuric and GlomerularDiseases

Assays for identifying at least one therapeutic agent for preventing ortreating recurrent proteinuric glomerular disease in at least onesubject (e.g., a plurality of subjects) are described herein. One assayfor identifying at least one therapeutic agent for preventing ortreating recurrent proteinuric glomerular disease in at least onesubject includes first contacting reporter cells with at least one of: aproteinuric glomerular disease-specific pool of sera from at least onesubject having the proteinuric glomerular disease, sera from a subjectwho does not have the proteinuric glomerular disease, and a chemicalthat either induces or reduces SMPDL-3b expression in at least onemulti-well plate. The reporter cells typically include a vector having anucleic acid encoding SMPDL-3b operably linked to a nucleic acidencoding a reporter (e.g., luciferase, GFP, etc.). The first portion ofthe reporter cells are subsequently or concomitantly contacted with(combined with, mixed with) a library of candidate therapeutic agents,while at least a second portion of the reporter cells is not contactedwith the library of candidate therapeutic agents. Reporter geneexpression is analyzed in the first and second portions of reportercells. By comparing reporter gene expression in the at least firstportion of reporter cells with reporter gene expression in the at leastsecond portion of cells, at least one therapeutic agent (e.g., 1, 2, 3,4, 5, 10, 50, etc.) from the library of candidate therapeutic agents isidentified that prevents modulation of SMPDL-3b expression and/orrestores SMPDL-3b expression to a control level. The therapeutic agent'sability to prevent modulation of SMPDL-3b expression or restore SMPDL-3bexpression relative to the control level is correlated with an abilityto prevent or treat recurrent proteinuric glomerular disease in the atleast one subject. Any suitable controls can be used for comparisons. By‘control level’ is typically meant podocyte specific physiologicalSMPDL-3b expression. Such an assay can further include examining atleast one culture of podocytes (e.g., normal human podocytes) in whichcytoskeletal rearrangements and/or disruptions have been induced by serafrom a subject having the proteinuric glomerular disease, or a chemicalthat induces cytoskeletal rearrangements. For example, the assay canfurther include correlating the therapeutic agent's ability to preventmodulation of SMPDL-3b expression or restore SMPDL-3b expression to thecontrol level with an ability to prevent or reverse cytoskeletaldisruptions or rearrangements observed in at least one culture ofpodocytes cultured in at least one multi-well plate with sera from atleast one subject having the proteinuric glomerular disease, or achemical that induces cytoskeletal rearrangements (e.g., cytokalasin D,protamine sulphate, lipopolysaccaride, puromycin aminonucleoside,phalloidin, and latrunculin). The podocytes can be analyzed forexpression of one or more factors that affect podocyte function, e.g.,SMPDL-3b, ASMase, ceramide, S1P, ABCA1, ABCG1, LDL-rec, ACC1, fatty acidsynthase, HMG-CoA reductase, and an SERBP.

Another assay for identifying at least one therapeutic agent forpreventing or treating recurrent proteinuric or glomerular disease in atleast one subject includes first culturing human podocytes in thepresence of at least one of: a proteinuric glomerular disease-specificpool of sera from at least one subject having the proteinuric glomerulardisease, sera from a subject who does not have the proteinuricglomerular disease, and a chemical that either induces or reducesSMPDL-3b expression in at least one multi-well plate. The culturedpodocytes are subsequently or concomitantly contacted with one or morecandidate therapeutic agents from a library of candidate therapeuticagents. Next, the cultured podocytes contacted with one or morecandidate therapeutic agents from a library of candidate therapeuticagents are examined for cytoskeletal disruptions or rearrangements. Byexamining the cultured podocytes for a decrease (relative to one or morecontrols) in cytoskeletal disruptions or rearrangements after exposureto a candidate therapeutic agent, at least one therapeutic agent whichprevents or decreases cytoskeletal disruptions or rearrangements can beidentified.

Methods and platforms for screening molecules, compounds, etc., forproteinuric and glomerular disease (e.g., FSGS) therapeutics caninclude, for example, small molecule screening and other compoundscreening through any Drug Discovery Core Facility. One example of amethod of identifying a therapeutic agent for preventing or treatingrecurrent proteinuric and glomerular disease (e.g., FSGS) in at leastone subject includes the steps of: culturing human podocytes in thepresence of a biological sample from at least one subject with high riskfor recurrent proteinuric and glomerular disease (e.g., FSGS) (asdetermined as described above, i.e., the subject is suspected ofdeveloping or is at high risk of developing proteinuric and glomerulardisease (e.g., FSGS) after receiving a kidney transplant when certainrisk factors are present, primarily age of the patient (less than 15years of age) and time from diagnosis to ESRD (less than 3 years));contacting the podocytes with one or more candidate therapeutic agents;examining the podocytes for cytoskeletal disruptions; and identifyingagents which prevent or decrease cytoskeletal disruptions. Examples ofcytoskeletal disruptions include disruption of stress fibers, or any ofthe read outs for specific podocyte proteins as described above (e.g.,the disruption of podocyte specific proteins involved in actincytoskeleton remodeling (synaptopodin) or the translocation of relevantplasma membrane proteins (nephrin, podocin, CD2AP) from the plasmamembrane (physiologic) to the cytosol (pathologic)). Agents thatstabilize podocyte cell membranes and prevent their degradation can beused to inhibit podocyte cytoskeletal disruptions in a human. An exampleof such an agent is one that modulates the activity and/or expression ofSMPDL-3b (sphingomyelin-phosphodiesterase-acid-like-3b), such asRituximab (Perosa F, Favoino E, Caragnano M A, Dammacco F., Blood 2006;107(3):1070-7). Another example of a drug that may be used to inhibitpodocyte cytoskeletal disruptions is abatacept (Orencia®, Bristol-MyersSquibb). In the examples below, clinical tests are described in whichabatacept reversed early post-transplant recurrent proteinuria in atleast one patient with previous recurrent FSGS (FIGS. 4A-4C).

In one embodiment, a method of identifying a therapeutic agent forpreventing or treating recurrent FSGS, DN or any other proteinuricglomerular disorder (FSGS, DN, Membranous nephropathy, minimal changedisease, IgA nephropathy, Membranoproliferative glomerulopathy, lupusnephritis, myeloma kidney, hypertensive nephrosclerosis, paucimmuneglomerulonephritis, preeclampsia, amyloidosis, cryoglobulinemia,thrombotic thrombocytopenic purpura, Hemolytic uremic syndrome,scleroderma kidney, Alport's glomerulopathy, storage disorders and otherrate genetic disorders) includes: culturing human podocytes in thepresence of a biological sample from at least one subject with thedisease; contacting the podocytes with one or more candidate therapeuticagents; examining the podocytes for cytoskeletal disruptions, excessiveor defective SMPDL-3b expression, lipid content, oxygen consumptionrate, apoptosis; and identifying agents which prevent or decreasecytoskeletal disruptions, maintain a physiological SMPDL-3b expression,reduce lipid content, reduce oxygen consumption rate, and reducesapoptosis. In the assay, modulation of SMPDL-3b can be analyzed by anysuitable methods, e.g., Western blotting, PCR, a SMP luciferase reporteractivity assay, etc. Lipid content can be measured by specific enzymaticreactions and by mass spectroscopy, oxygen consumption rate by seahorsetechnology and apoptosis by ELISA or flow cytometry. The steps ofcontacting the podocytes with one or more candidate therapeutic agents,examining the podocytes for cytoskeletal disruptions, SMPDL-3bexpression, lipid content, oxygen consumption rate, apoptosis; andidentifying agents which prevent or decrease cytoskeletal disruptions,restores physiological SMPDL-3b expression, lipid content, oxygenconsumption rate, apoptosis can include (but it is not limited to)screening a library of candidate therapeutic agents (e.g., in ahigh-throughput multi-well format). The candidate therapeutic agents maybe small molecules or any currently approved medication for eachindividual condition or any off label therapeutic agent.

One example of a method of identifying a therapeutic agent forpreventing or treating proteinuric and glomerular disease (e.g., FSGS)in humans includes screening a library of potential therapeutic agents(e.g., small molecules) to identify one or more agents that inhibitpodocyte cytoskeletal disruptions in a human. In a typical embodiment,the library is screened in a high-throughput multi-well format. Thecandidate therapeutic agents (e.g., compounds, candidate therapeuticagents, candidate agents, test compounds) can be any organic, inorganic,small molecule, protein, antibody, aptamer, nucleic acid molecule, orsynthetic compound. Candidate compounds identified by assays describedherein as useful pharmacological agents can be pharmacological agentsalready known in the art or variations thereof or can be compoundspreviously unknown to have any pharmacological activity. The candidatecompounds can be naturally occurring or designed in the laboratory.Candidate compounds can comprise a single diastereomer, more than onediastereomer, or a single enantiomer, or more than one enantiomer.

Candidate or potential therapeutic agents may be obtained from a widevariety of sources including libraries of synthetic or naturalcompounds. For example, numerous means are available for random anddirected synthesis of a wide variety of organic compounds andbiomolecules, including expression of randomized oligonucleotides.Alternatively, libraries of natural compounds in the form of e.g.bacterial, fungal and animal extracts are available or readily produced.The candidate or potential therapeutic agents can be obtained using anyof the numerous approaches in combinatorial library methods known in theart, including: biological libraries; peptoid libraries (libraries ofmolecules having the functionalities of peptides, but with a novel,non-peptide backbone which are resistant to enzymatic degradation butwhich nevertheless remain bioactive); spatially addressable parallelsolid phase or solution phase libraries; synthetic library methodsrequiring deconvolution; the one-bead one-compound library method; andsynthetic library methods using affinity chromatography selection.Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner,U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. 5,223,409),plasmids (Cull et al. (1992) Proc Nat'l Acad Sci USA 89:1865-1869) or onphage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382;Felici (1991) J. Mol. Biol. 222:301-310).

One or more systems, methods or both can be used to identify a candidateor potential therapeutic agent for proteinuric and glomerular disease(e.g., FSGS). Manual systems/methods, semi-automated systems/methods,and automated systems/methods are all possible. A variety of robotic orautomatic systems are available for automatically or programmablyproviding predetermined motions for handling, contacting, dispensing, orotherwise manipulating materials in solid, fluid liquid or gas formaccording to a predetermined protocol. Such systems may be adapted oraugmented to include a variety of hardware, software or both to assistthe systems in determining mechanical properties of materials. Hardwareand software for augmenting the robotic systems may include, but are notlimited to, sensors, transducers, data acquisition and manipulationhardware, data acquisition and manipulation software and the like.Exemplary robotic systems are commercially available from CAVROScientific Instruments (e.g., Model NO. RSP9652) or BioDot (MicrodropModel 3000).

Assays for Predicting if a Diabetic Subject Will Develop Kidney Disease

The assays described herein for predicting risk of primary or recurrentproteinuric glomerular disease (e.g., FSGS) can be modified as neededand used to predict if a diabetic subject will develop kidney disease. Atypical assay for predicting if at least one diabetic subject willdevelop kidney disease includes: contacting a biological sample from theat least one diabetic subject with a first culture of human podocytes;examining the first culture of human podocytes for modulation (e.g.,upregulation, downregulation, degradation of) of SMPDL-3b and/or anyrelated lipid component, and/or particular cellular abnormalities suchas cellular enlargement, cellular blebbing, loss of stress fibers,oxygen consumption rate and apoptosis; and correlating a presence ofparticular cellular abnormalities (e.g., cellular enlargement, cellularblebbing, loss of stress fibers, lipid accumulation, oxygen consumptionrate and apoptosis) with an increased risk of development of kidneydisease in the subject compared to a non-diabetic subject or a subjecthaving diabetes without the kidney disease. Generally, the step ofexamining the first culture of human podocytes for particular cellularabnormalities (e.g., cellular enlargement, cellular blebbing, loss ofstress fibers, lipid accumulation, oxygen consumption rate andapoptosis) includes determining a cytoskeletal phenotype of the firstculture of human podocytes. This cytoskeletal phenotype can be comparedto a cytoskeletal phenotype of a second culture of human podocytes thatwere contacted with a biological sample from at least one non-diabeticsubject, or at least one diabetic subject who does not have the kidneydisease (a negative control). The step of examining the first culture ofhuman podocytes for cellular abnormalities (e.g., cellular enlargement,cellular blebbing and loss of stress fibers) is typically performedusing confocal or light microscopy. However, any suitable device and/ormeans of examining the human podocytes for cellular abnormalities can beused. A disease-specific podocyte phenotype could be identified with theproposed assay for any other glomerular disorder (including those listedherein), and a specific prediction assay could be developed for eachlocal or systemic disease.

Kits

A plurality of kits for performing assays and delivering treatments aredescribed herein. A kit for determining if at least one subject who isat risk for a primary or secondary proteinuric glomerular disorder orwho has a primary or secondary proteinuric glomerular disorder is atrisk for primary or secondary disease development, progression orrecurrence of the proteinuric glomerular disorder after kidneytransplantation includes a plurality of reporter cells comprising avector containing a nucleic acid encoding the SMPDL-3b promoter sequenceoperably linked to a nucleic acid encoding a reporter gene; at least onecontrol; and instructions for use. In an alternative embodiment, a kitfor determining if at least one subject who is at risk for a primary orsecondary proteinuric glomerular disorder or who has a primary orsecondary proteinuric glomerular disorder is at risk for primary orsecondary disease development, progression or recurrence of theproteinuric glomerular disorder after kidney transplantation includes atleast one container of podocytes; cytochalasin D as a positive control;a dye for actin cytoskeleton; and instructions for use. In addition toor alternatively to cytochalasin D, any suitable positive control can beused. Such positive controls are known in the art. A kit for predictinga response in at least one subject to at least one candidate therapeuticagent for treatment or prevention of a particular proteinuric glomerulardisease includes a plurality of reporter cells including a vectorcontaining a nucleic acid encoding the SMPDL-3b promoter sequenceoperably linked to a nucleic acid encoding a reporter gene; a pluralityof candidate therapeutic agents; at least one control; and instructionsfor use. In an alternative embodiment, a kit for predicting a responsein at least one subject to at least one candidate therapeutic agent fortreatment or prevention of a particular proteinuric or glomerulardisease includes at least one container of podocytes; cytochalasin D asa positive control; a dye for actin cytoskeleton; a plurality ofcandidate therapeutic agents; and instructions for use.

In some embodiments, kits are provided for determining if a subject isat risk for primary glomerulopathy, secondary glomerulopathy orpost-transplant recurrence of any glomerular disease (FSGS, DN,Membranous nephropathy, minimal change disease, IgA nephropathy,Membranoproliferative glomerulopathy, lupus nephritis, myeloma kidney,hypertensive nephrosclerosis, paucimmune glomerulonephritis,amyloidosis, cryoglobulinemia, thrombotic thrombocytopenic purpura,Hemolytic uremic syndrome, scleroderma kidney, Alport's glomerulopathy,transplant glomerulopathy, storage disorders and other rare geneticdisorders). Such kits can be used, for example, to determine if at leastone subject (e.g., human) who has FSGS is at risk for recurrent FSGSafter kidney transplantation. A typical kit for determining if at leastone subject (e.g., human) who has a proteinuric and glomerular disease(e.g., FSGS) is at risk for recurrent proteinuric and glomerular disease(e.g., recurrent FSGS) after kidney transplantation includes a pluralityof podocytes, at least one control (e.g., negative control, positivecontrol), and instructions for use. In one embodiment, a kit includes avial of podocytes, a set of Cytochalasin D vials at differentconcentrations to be utilized for a standard curve, and instructions foruse/protocols for treatment and media preparation.

In the kits, any suitable controls and reagents for generating standardcurves may be used. Cytochalasin D is just one example of a regent forgenerating a standard curve, and others may be used. Similarly, inaddition to a dye for actin cytoskeleton, any marker or label suitablefor examining or visualizing the actin cytoskeleton may be used (e.g.phalloidin, anti F actin antibodies). Any of the kits may include acollagen coated well plate to carry a mixture of the different reagents,as well as one or more washing buffers. Optionally, kits may alsocontain one or more of the following: containers which include positivecontrols, containers which include negative controls, photographs orimages of representative examples of positive results and photographs orimages of representative examples of negative results. Kits may alsoinclude reagents for the determination of podocyte lipid content,apoptosis, and oxygen consumption rate when considered appropriate for aspecific proteinuric glomerular disease.

Compositions and Methods for Preventing or Treating Proteinuric andGlomerular Diseases

Compositions and methods for preventing and/or treating proteinuric andglomerular diseases or disorders (e.g., FSGS, DN, Membranousnephropathy, minimal change disease, IgA nephropathy,Membranoproliferative glomerulopathy, lupus nephritis, myeloma kidney,hypertensive nephrosclerosis, paucimmune glomerulonephritis,preeclampsia, amyloidosis, cryoglobulinemia, thrombotic thrombocytopenicpurpura, Hemolytic uremic syndrome, scleroderma kidney, Alport'sglomerulopathy, storage disorders and other rate genetic disorders) in asubject are described herein. Using the screening assays and platformsdescribed herein, one or more therapeutic agents for preventing and/ortreating a proteinuric or glomerular disorder can be identified. In oneembodiment of a method of preventing and/or treating a proteinuric orglomerular disorder (e.g., recurrent FSGS) in a subject, the methodincludes providing a composition including an agent that restoresphysiological expression of SMPDL-3b, lipid content, oxygen consumptionrate or apoptosis, thus driving personalized therapeutic decisions aboutwhich composition to administer to the subject in a therapeuticallyeffective amount for preventing and/or treating the proteinuric orglomerular disorder (e.g., recurrent FSGS) in the subject. In oneexample, the agent that upregulates activity and/or expression ofSMPDL-3B is rituximab, a chimeric antibody directed against CD20 thathas been developed for the cure of lymphoma and that has been found tobind SMPDL-3B as well (Perosa F, Favoino E, Caragnano M A, Dammacco F.Blood 2006; 107(3):1070-7). A method of preventing and/or treatingrecurrent FSGS in a subject may include administering to the subject acomposition including an agent that increases SMPDL-3b levels orrestores cytoskeleton rearrangements and a pharmaceutically acceptablecarrier, wherein the composition is administered to the subject havingFSGS in an amount effective to prevent or treat FSGS in the subject. Forexample, a drug such as abatacept can be administered to the subject. InExample 4 below (FIGS. 4A-4C), clinical tests are described in whichabatacept reversed early post-transplant recurrent proteinuria in atleast one patient with previous recurrent FSGS.

The screening assays and platforms described herein may find particularuse in identifying therapeutic agents known for treating disorders otherthan proteinuric and glomerular disorders that are useful also forpreventing and/or treating a proteinuric or glomerular disorder. Suchoff label use of drugs (e.g., abatacept, rituximab) for treating aproteinuric or glomerular disorder is exemplified in the Examplessection below.

Compositions and Methods for Preventing and/or Treating Renal Diseasesthat Modify Cellular Lipid Content

Described herein are methods of administering cyclodextrin or itsderivatives, or any drug lowering the plasma membrane or cellularcholesterol/lipid content, to a patient for a time and under conditionssufficient to prevent, treat, cure, or reverse renal-related disorders.It is known that elevated cholesterol levels, and in particularlow-density lipoprotein (LDL) cholesterol, in the plasma play animportant role in the development of kidney disease and otherrenal-related diseases. Currently available treatments to lowercholesterol levels in patients, however, aim to lower plasma cholesterollevels (LDL) by blocking the synthesis of cholesterol in the liver(statins), by preventing reabsorption of cholesterol into thecirculatory system (bile acid resins, cholesterol absorptioninhibitors), or by increasing HDL cholesterol (fibrates, niacinderivatives). None of the currently used medications aims on loweringplasma membrane or cellular cholesterol/lipid.

A typical method of preventing or treating a renal-related disorder in asubject includes administering to the subject a composition includingone or more of: a cyclodextrin, a cyclodextrin derivative, and acellular cholesterol-lowering agent that is not a statin (alone or incombination with other drugs currently used to treat the subject for arenal-related disorder). The composition is administered to the subjectin an amount effective for reducing one or more of: plasma membranecholesterol, plasma membrane lipids, cellular cholesterol, and cellularlipids in the subject. Typically, the composition is administered in anamount effective for preserving podocyte function and preventingproteinuria in the subject. Additionally, administration of thecomposition may result in reduction of plasma membrane cholesterol,cellular cholesterol, or cellular lipids in podocytes of the kidney ofthe subject. Examples of renal-related diseases include primaryglomerulopathy, secondary glomerulopathy, or a post-transplantrecurrence of a glomerular disease (e.g., FSGS, Membranous nephropathy,minimal change disease, IgA nephropathy, Membranoproliferativeglomerulopathy, diabetic nephropathy, lupus nephritis, myeloma kidney,hypertensive nephrosclerosis, paucimmune glomerulonephritis,preeclampsia, amyloidosis, cryoglobulinemia, thrombotic thrombocytopenicpurpura, Hemolytic uremic syndrome, scleroderma kidney, Alport'sglomerulopathy, transplant glomerulopathy storage disorder, stroke,peripheral vascular disease, diabetes, coronary artery disease,congestive heart failure, cardiac hypertrophy, myocardial infarction,endothelial dysfunction and hypertension). In the method, thecomposition can further include a drug such as: an immunosuppressiveagent, an ACTH agonist, an insulin sensitizer, a GH antagonist, anantinflammatory medication, a vitamin D derivative, a RAS systeminhibitor, an aldosterone inhibitor, an HMG-CoA reductase inhibitor, acholesterol absorption inhibitor, a bile acid sequestrant, a bile acidresin, Niacin, a Niacin derivative, a fibrate, a cholesteryl estertransfer protein (CETP) inhibitor, an Acetyl-Coenzyme Aacetyltransferase (ACAT) inhibitor, and a microsomal triglyceridetransport inhibitor.

One embodiment of a method of treatment relates to renal-relateddisorders such as proteinuric diseases, albuminuric diseases, Diabeticnephropathy, Nephrotic or Nephritic syndromes, Toxic lesions of thekidneys, glomerular diseases such as FSGS, IgA or IgM nephropathy,Membranoproliferative glomerulonephritis, Membranous nephropathy,Minimal change disease, Hypertensive nephrosclerosis or Interstitialnephritis. In another embodiment, a renal-related disorder can be, forexample, stroke, peripheral vascular disease, coronary artery diseases,congestive heart failure, cardiac hypertrophy, myocardial infarction,endothelial dysfunction and hypertension. In a typical embodiment, thedrug used to prevent, treat, cure or reverse renal-related disorders isany drug that lowers the plasma membrane and cellular cholesterol/lipidcontent of the cell. The drug can hereby be administered to anindividual in a variety of ways. Routes of administration include,intramuscular, intraperitoneal, intravenous (systemic), subcutaneous,transdermal, oral, topical, and intranasal routes. The drug can beadministered together with other biologically active agents orcomponents as pharmaceutically acceptable carriers, diluents, excipientsand vehicles. In one embodiment, the drug used to prevent, treat, cureor reverse renal-related disorders is Cyclodextrin or any of itsderivatives. In another embodiment, Cyclodextrin is used to prevent,treat or reduce proteinuria in patients.

Also described herein are methods of reducing the plasma membrane orcellular cholesterol/lipid content in any cells of any organ as a toolto prevent, treat, cure or reverse renal-related disorders. In oneembodiment, the plasma membrane or cellular cholesterol/lipid content isreduced in any cell of the kidney as a tool to prevent, treat, cure orreverse renal-related disorders. In another embodiment, the plasmamembrane or cellular cholesterol/lipid content is reduced in podocytesof the kidney as a tool to prevent, treat, cure or reverse renal-relateddisorders. In another embodiment, Cyclodextrin or its derivatives areused to at least partially deplete kidney cells from cholesterol/lipidalone or in combination with other drugs currently used or being studiedfor the prevention and the treatment of kidney-related diseases such asimmunosuppressive agents, ACTH agonists, insulin sensitizers, GHantagonists, antinflammatory medications, vitamin D derivatives,blockers of the RAS system and of aldosterone. In another embodiment,Cyclodextrin, its derivatives, or any other plasma membrane or cellularcholesterol/lipid lowering drug is used in combination with acholesterol biosynthesis inhibitor, such as a HMG-CoA reductaseinhibitor. HMG-CoA reductase inhibitor drugs include drugs such asSimvastatin, Atorvastatin, Lovastatin, Rosuvastatin, Pravastatin,Fluvastatin, Pitavastatin, Rosuvastatin, Rivastatin, Itavastatin, orZD-4522. In another embodiment, Cyclodextrin, its derivatives, or anyother plasma membrane or cellular cholesterol lowering drug is used incombination with a cholesterol absorption inhibitor, such as aEzetimibe, SCH-48461. In another embodiment, Cyclodextrin, itsderivatives, or any other plasma membrane or cellular cholesterol/lipidlowering drug is used in combination with bile acid sequestrants andresins (Colestipol, Colestilan, Colextran, Cholestyramine, Colesevelam).In another embodiment, Cyclodextrin, its derivatives, or any otherplasma membrane or cellular cholesterol/lipid lowering drug is used incombination with Niacin and Niacin derivatives such as Niceritrol,Nicotinyl alcohol, Acipimox. In another embodiment, Cyclodextrin, itsderivatives, or any other plasma membrane or cellular cholesterol/lipidlowering drug is used in combination with fibrates such as Fenobrate,Clinofibrate, Etofibrate, Bezafibrate, Gemfibrozil. In anotherembodiment, Cyclodextrin, its derivatives, or any other plasma membraneor cellular cholesterol/lipid lowering drug is used in combination withcholesteryl ester transfer protein (CETP) inhibitors such asDalcetrapib, Anacetrapib. In another embodiment, Cyclodextrin, itsderivatives, or any other plasma membrane or cellular cholesterol/lipidlowering drug is used in combination with Acetyl-Coenzyme Aacetyltransferase (ACAT) inhibitors (such as avasimibe) or microsomaltriglyceride transport inhibitors.

These combination treatments may also be effective for the treatment orcontrol of one or more renal-related conditions such as atherosclerosis,insulin resistance, hyperlipidemia, hypertriglyceridemia, dyslipidemia,high LDL, and low HDL.

Data and Analysis

Use of the assays, methods and kits described herein may employconventional biology methods, software and systems. Useful computersoftware products typically include computer readable medium havingcomputer-executable instructions for performing logic steps of a method.Suitable computer readable medium include floppy disk,CD-ROM/DVD/DVD-ROM, hard-disk drive, flash memory, ROM/RAM, magnetictapes and etc. The computer executable instructions may be written in asuitable computer language or combination of several languages. Basiccomputational biology methods are described in, for example Setubal andMeidanis et al., Introduction to Computational Biology Methods (PWSPublishing Company, Boston, 1997); Salzberg, Searles, Kasif, (Ed.),Computational Methods in Molecular Biology, (Elsevier, Amsterdam, 1998);Rashidi and Buehler, Bioinformatics Basics: Application in BiologicalScience and Medicine (CRC Press, London, 2000) and Ouelette and BzevanisBioinformatics: A Practical Guide for Analysis of Gene and Proteins(Wiley & Sons, Inc., 2nd ed., 2001). See U.S. Pat. No. 6,420,108.

The assays, methods and kits described herein may also make use ofvarious computer program products and software for a variety ofpurposes, such as reagent design, management of data, analysis, andinstrument operation. See, U.S. Pat. Nos. 5,593,839, 5,795,716,5,733,729, 5,974,164, 6,066,454, 6,090,555, 6,185,561, 6,188,783,6,223,127, 6,229,911 and 6,308,170. Additionally, the embodimentsdescribed herein include methods for providing data (e.g., experimentalresults, analyses) and other types of information over networks such asthe Internet.

ADDITIONAL EMBODIMENTS

It should be clear from the present disclosure that the assays and kitsdescribed herein include screening individuals without kidney diseasefor disease development, as well as screening individuals with kidneydisease for disease progression or recurrence after transplant (e.g.,kidney transplant). Such screening can be done using the protocols,methods, reagents, kits and assays described herein.

An assay for determining if at least one subject who has FSGS is at riskfor recurrent FSGS after kidney transplantation may include: contactinga biological sample from the at least one subject with a culture ofhuman podocytes; examining the podocytes for the presence or absence ofcytoskeletal disruptions or rearrangements, lipid accumulation,modulation of physiological SMPDL-3b levels, and or apoptosis resultingin determination of a first phenotype of the podocytes; and correlatinga presence of cytoskeletal disruptions or rearrangements of stressfibers with an increased risk of FSGS recurring in the subjectsubsequent to a kidney transplantation compared to subjects withoutrecurrent FSGS. In the assay, the biological sample (e.g., blood,saliva, serum, plasma, tissue, and urine) can be obtained prior to thekidney transplantation and initiation of immunosuppression in thesubject. The cytoskeletal disruptions or rearrangements (e.g.,disruption of stress fibers) can be examined by the methods describedherein. The assay can further include comparing the cytoskeletalphenotype of human podocytes with the quantitative determination ofcholesterol/lipid accumulation, modulation of SMPDL-3b, and/orapoptosis. In some embodiments, the subject has an increased risk ofFSGS recurring subsequent to a kidney transplantation compared tosubjects without recurrent FSGS, and the assay further includes making aquantitative assessment of an FSGS phenotype. The assay can furtherinclude comparing the first phenotype with a second phenotype ofpodocytes contacted with a biological sample from at least one normalsubject, and a third phenotype of podocytes contacted with a biologicalsample from at least one subject with recurrent FSGS. The secondphenotype is typically a negative control, and the third phenotype apositive control. The at least one subject (e.g., pediatric and/orAfrican American) can be a plurality of subjects who have FSGS. In someassays, a standard curve is analyzed. The step of examining thepodocytes for the presence or absence of cytoskeletal disruptions orrearrangements resulting in determination of a first phenotype of thepodocytes can include counting the number of podocytes that displaycytoskeletal disruptions or rearrangements. Generally, at least onesubject who is at risk for development of recurrent FSGS is identifiedprior to the at least one subject receiving a kidney transplant.

An assay for predicting if at least one diabetic subject will developkidney disease (e.g., diabetic nephropathy, proteinuric kidney disease,etc.) includes: contacting a biological sample from the at least onediabetic subject with a first culture of human podocytes; examining thefirst culture of human podocytes for cellular enlargement, cellularblebbing, cortical distribution of the actin cytoskeleton stress fibers,modulation of SMPDL-3b, lipid accumulation, oxygen consumption rate andapoptosis; and correlating the presence of one or more of: cellularenlargement, cellular blebbing and cortical distribution of the actincytoskeleton stress fibers, modulation of physiological SMPDL-3b levels,cholesterol/lipid accumulation, increased oxygen consumption rate andapoptosis with an increased risk of development of kidney disease in thesubject compared to a non-diabetic subject or a subject having diabeteswithout the kidney disease. The step of examining the first culture ofhuman podocytes for cellular enlargement, cellular blebbing, loss ofstress fibers can include determining a cytoskeletal phenotype of thefirst culture of human podocytes. The assay can further includecomparing the cytoskeletal phenotype of the first culture of humanpodocytes with a cytoskeletal phenotype of a second culture of humanpodocytes that were contacted with a biological sample from at least onenon-diabetic subject, or at least one diabetic subject who does not havethe kidney disease. The assay can further include comparing thecytoskeletal phenotype of human podocytes with the quantitativedetermination of cholesterol/lipid accumulation, modulation ofphysiological SMPDL-3b levels, increased oxygen consumption rate andapoptosis. The step of examining the first culture of human podocytesfor cellular enlargement, cellular blebbing and cortical distribution ofthe actin cytoskeleton stress fibers can be performed using any suitablemethod, e.g., confocal or light microscopy.

A method of identifying a therapeutic agent for preventing or treatingrecurrent FSGS in at least one subject includes: culturing humanpodocytes in the presence of a biological sample from at least onesubject with recurrent FSGS; contacting the podocytes with one or morecandidate therapeutic agents; examining the podocytes for cytoskeletaldisruptions; and identifying agents which prevent or decreasecytoskeletal disruptions. The steps of contacting the podocytes with oneor more candidate therapeutic agents, examining the podocytes forcytoskeletal disruptions; and identifying agents which prevent ordecrease cytoskeletal disruptions can include screening a library ofcandidate therapeutic agents (e.g., in a high-throughput multi-wellformat). The candidate therapeutic agents may be small molecules or anycurrently approved medication for each individual condition. Thecandidate therapeutic agents can also be off label drugs. The methoddescribed herein may therefore allow for the identification ofpersonalized treatment strategies for any patient with any renaldisorder.

A method of preventing or treating recurrent FSGS in a subject includesthe steps of: providing a composition including an agent thatupregulates activity and/or expression of SMPDL-3b; and administeringthe composition to the subject in a therapeutically effective amount forpreventing recurrent FSGS in the subject. The agent can be, for example,an antibody that stabilizes cellular membranes and prevents degradationof cellular membranes. Additional examples of agents include nucleicacid, proteins, polypeptides, compounds, off-label drugs, extracts,cells, etc. A method for preventing or treating DN may involve agentsthat affect podocyte function through the modulation of cellularcholesterol/lipid content (e.g., sterols, sphingolypids, triglyceride,free fatty acids, glycosphingolypids, ceramide). Therefore, any agentcapable of modulating podocyte SMPD1-3b or any lipid related protein(ASMase, S1P, ABCA1, ABCG1, LDL-rec, ACC1, fatty acid synthase,stearoyil-CoA desaturase, HMG-CoA reductase, SERBPs) can be utilized forthe prevention and the cure of DN and of any other proteinuric renaldisorder.

EXAMPLES

The present invention is further illustrated by the following specificexamples. The examples are provided for illustration only and should notbe construed as limiting the scope of the invention in any way.

Example 1—Assay for the Prediction of Recurrent FSGS after KidneyTransplantation

Shown in FIG. 1A are representative stress fiber confocal images ofnormal human podocytes exposed to normal (NHS) (n=5), non-recurrent(NON-REC) FSGS (n=10), and recurrent (REC) FSGS (n=12) human sera. Shownin FIG. 1B is the percentage of cells with disruption of stress fibersobserved after exposure to NHS (n=5), non-recurrent sera (n=10), andrecurrent human sera (n=12). FIG. 1C shows the linear correlationbetween the percentage of cells with loss of stress fibers and the urineprotein/creatinine ratio obtained from REC and NON-REC patients (n=22)in the first 30 days after transplantation. Shown in FIG. 1D areconfocal images of stress fibers and corresponding bar graph analysis ofnormal human podocytes exposed to recurrent FSGS sera in the presence(REC+RITUX) or absence (REC) of rituximab. Rituximab protected the lossof stress fibers observed in stressed podocytes exposed to recurrentFSGS, but not non-recurrent FSGS human sera. Shown in FIG. 1E areconfocal images of stress fibers and corresponding bar graph analysis ofnormal human podocytes exposed to REC sera transfected with an empty GFPvector (REC) or with a SMPDL-3b-GFP vector (REC+SMPDL-3b). SMPDL-3boverexpression protected the loss of stress fibers observed in podocytesexposed to recurrent FSGS human sera. The presented in vitro studiesstrongly correlate with in vivo clinical outcome data, as rituximabadministered in patients at high risk for recurrent FSGS aftertransplantation significantly protected from the development of posttransplant proteinuria as demonstrated in Table 1.

TABLE 1 Patient demographics and clinical outcome. Controls (Norituximab) Treated (Rituximab) N = 14 N = 27 p value Age (mean ± SD)12.3 ± 5.2  15.0 ± 5.5  0.1650 Race (W/B) 9/5 (64%/36%) 14/13 (52%/48%)0.5203 Gender (M/F) 6/8 (43%/57%)  9/18 (33%/67%) 0.7337 Time to ESRD(year) 3.3 ± 2.1 3.4 ± 2.0 0.7942 Donor (LD/DD) 9/5 (64%/36%)  4/23(15%/85%) 0.0033* Donor age (mean ± SD) 31.3 ± 9.4  24.7 ± 14.6 0.1290Nephrectomy (Y/N) 7/7 (50%/50%) 16/11 (59%/41%) 0.7417 Nephroticproteinuria w/i 1 Mo  9 (64%) 7 (26%) 0.229* Plasmapheresis w/I 1 Mo 10(71%) 8 (30%) 0.0192* CD 19 count Week 0 412 ± 223 360 ± 223 0.4344 Week0.5 327 ± 290 107 ± 115 0.0145* Week 1 472 ± 437 45 ± 31 <0.0001* Week 2559 ± 526 16 ± 23 <0.0001* Week 3 729 ± 670 5 ± 6 <0.0001* Week 4 630 ±384 6 ± 7 0.0002* ΔeGFR (vs. 1 mo nadir)  3 months −18.0 ± 16.9  −1.3 ±14.6 0.0012*  6 months −19.0 ± 19.8  −5.3 ± 18.4 0.0075* 12 months −26.9± 26.7  −20.3 ± 27.3  0.3717 Graft survival  6 months 92.9%  100% 0.173012 months 85.7% 95.8% 0.2639

Age, race, gender, nephrectomy of the native kidneys, time to ESRD anddonor characteristics are shown for the 14 historical control patientscompared to the 27 patients that received one dose of rituximab (375mg/m²) within 24 hours of transplant. CD19+ cells significantlydecreased in treated patients 0.5 weeks after infusion and were almostundetectable 1 week after treatment. The incidence of recurrentnephrotic range proteinuria and need for plasmapheresis between day 3and 30 after transplantation was significantly lower in the rituximabgroup than in the control group. The changes of estimated GFR (ΔeGFR) at3-6 months from baseline at 1 month post-transplant were significantlyworse in control group than rituximab group. There was no significantdifference in graft survival between groups at 6 and 12 months. W=white,B=black, M=male, F=female, ESRD=end stage renal disease, LD=livingdonor, DD=deceased donor. ΔeGFR: variation of eGFR. *p<0.05.

Example 2—Rituximab and/or SMPDL-3b Upregulation has a Protective Effectin FSGS

FIGS. 2A-2G demonstrate that the modulation of SMPDL-3b in kidneybiopsies and in podocytes cultured in the presence of patient sera canbe predictive of recurrence of FSGS after transplantation. (A) Low- andhigh-power images of immunoperoxidase staining for SMPDL-3b andsynaptopodin in post-reperfusion biopsies of patients with recurrent(REC) and non-recurrent (NON-REC) FSGS. Arrows point to podocytes. (B)The number of SMPDL-3b+ podocytes per glomerulus, as evaluated bySMPDL-3b and synaptopodin labeling in post-reperfusion kidney biopsiesfrom patients that later on developed recurrent (REC) disease (n=8) andpatients that did not develop clinical recurrence (NON-REC) (n=12). Allkidney biopsies were obtained prior to initiation of treatment withrituximab. (C) Regulation of podocyte SMPDL-3b mRNA expression by normal(NHS), non-recurrent (NON-REC) FSGS, and recurrent (REC) FSGS human sera(n=4 per group) and by rituximab. (D) The amount of SMPDL-3b protein isnormalized to actin in human podocytes treated with normal (n=5),recurrent (n=12), or non-recurrent (n=10) human sera and exposed torituximab. (E) Western blot for SMPDL-3b protein of normal podocytescultured with sera from consecutive non-recurrent (n=4) and recurrent(n=4) FSGS patients in the presence or absence of rituximab. (F) Theamount of 52 and 54 kDa ASMase protein is normalized to actin in humanpodocytes that were exposed to normal (n=5), non-recurrent FSGS (n=10),and recurrent FSGS (n=12) human sera in the presence or absence ofrituximab. (G) ASMase activity per μg of total lysate protein, asevaluated by ELISA.

Referring to FIGS. 2A-2G and Table 1, experiments were performed thatdemonstrate that rituximab and SMPDL-3b overexpression equally protectpodocytes from REC sera-induced disruption of stress fibers. Rituximab(Perosa F, Favoino E, Caragnano M A, Dammacco F. Blood 2006;107(3):1070-7) is a chimeric antibody directed against CD20 that hasbeen developed for the cure of lymphoma and that has been found to bindSMPDL-3B as well (Perosa F, Favoino E, Caragnano M A, Dammacco F. Blood2006; 107(3):1070-7). Therefore, the assay proposed herein could beutilized not solely as a prediction assay for recurrent disease but alsoas a pre-transplant assay to determine which drug, if any, would be moreefficient in preventing recurrent proteinuria in a given patient. It isexpected that drugs administered to podocytes in vitro prior or aftersera exposure may protect stress fibers formation, similar to what hasbeen shown for Rituximab in FIGS. 1A-1E. This assay could thereforeoffer a pre-transplant measure of the ability of a given patient torespond to a given drug after transplantation.

Example 3—Assay for the Prediction of Diabetic Nephropathy

DN is a chronic progressive disease that affects a sizable subset ofpatients with diabetes. Despite the identification of multiple riskfactors that contribute to the development of DN, it has remaineddifficult to identify the subset of patients with diabetes at risk forDN. This led to testing to determine if an in vitro prediction assaycould be developed. For this purpose, the following experiments wereconducted. Non-identifiable samples of patient sera were obtained.Samples consisted of serum collected from 10 patients with diabetes andmacroalbuminuria (DM w DN) and 10 diabetic normoalbuminuric patients (DMw/o DN) well matched for HgA1_(C) (7.9%), total cholesterol levels andthe duration of diabetes. Normal human sera (NHS) from 10 age and sexmatched healthy subjects were also utilized as controls. Differentiatedhuman podocytes were serum starved for 24 hours and then exposed to 4%serum of NHS, DM w/o DN, DM w DN for 24 hours. After a brief fixationwith 4% PFA, immunostainings for F-actin and DAPI were performed anddemonstrated that DM w DN sera treated podocytes experienced apronounced actin cytoskeleton meshwork with localized decoupling of thecytoskeleton from the plasma membrane (blebbing), which was evident inboth, the phalloidin staining as well as in the brightfield images (FIG.3A). Quantitative analysis of the cell blebbing (percentage of cellswith blebs out of a total of 200 cells analyzed) revealed this phenotypein 80% of cells exposed to sera of the DM w DN group as compared to only20% in the cells exposed to DM w/o DN and 5% in the NHS (FIG. 3B,p<0.01). Although filipin staining revealed increased cellularcholesterol in cells exposed to both DM w/o DN and DM w DN sera, thiswas particularly pronounced in the DM w DN group, where it wasaccompanied by a redistribution of phosphorylated caveolin (utilized asa marker of focal adhesion contact) to blebs, a phenomenon that could beprevented by cyclodextrin (CD) (FIG. 3C). Total cholesterol was alsodetermined with an enzymatic reaction. Total cholesterol was increasedin podocytes treated with DM w DN when compared to DM w/o DN and NHS(FIG. 3D, *p<0.05), a phenomenon that was prevented by CD (FIG. 3D, #p<0.05). Cell blebbing was predictive of DN, as sera collected frompatients before progressing to DN at the time they were normoalbuminuricalready caused cell blebbing (FIG. 3E). DM w DN sera also resulted inincreased podocyte apoptosis (FIG. 3F, *p<0.05, **p<0.01), a phenomenonthat could be prevented by CD (FIG. 3F, #p<0.05). Taken together, thesedata suggest that it is possible to develop an in vitro assay to screenpatients with diabetes for their risk to develop DN.

Example 4—Use of Abatacept to Reverse Early Post-Transplant RecurrentProteinuria in Patients with Established Recurrent FSGS

Described herein for the first time, is the successful use of abataceptand plasmapheresis to reverse post-transplant early recurrentproteinuria in two patients with a second allograft with a history ofprevious recurrent FSGS. A post-reperfusion kidney biopsy demonstrateddamage to podocytes (electron microscopy) not present on thepre-perfusion biopsy. The response to abatacept was associated with thepodocyte specific appearance of B7-1/CD80 expression on thepost-reperfusion renal biopsy when compared to the pre-perfusion biopsy,similar to what has been reported in patients with proliferative lupusnephritis (FIGS. 4A-4C). This case report highlights the possible roleof B7-1/CD80 in the pathogenesis of recurrent FSGS and offers a novelrationale for the utilization of Anti-B7-1/CD80 therapy in this setting.

Case Report 1

A 28-year old woman who was diagnosed with FSGS with mesangialproliferation presented with nephrotic range proteinuria at the age of16 years. Despite aggressive treatment with high dose steroids,tacrolimus, angiotensin converting enzyme inhibitor and angiotensin-2receptor blocking therapy, she developed ESRD within 7 years of FSGSdiagnosis. While she was on haemodialysis, she underwent medicalnephrectomy by using high-dose nonsteroidal anti-inflammatory agents toreduce her severe proteinuria. Two months after starting dialysis, sheunderwent one-haplotype-matched living related renal transplant at 23years old from her 53-year-old father. She received Thymoglobulin (1mg/kg×5 doses), daclizumab (1 mg/kg×2 doses) and methylprednisolone asinduction therapy, 1 dose of rituximab (375 mg/m²) and plasmapheresis.Renal function recovered after transplantation with mild postoperativeproteinuria that responded to plasmapheresis. The immunosuppression wasmaintained with tacrolimus, mycophenolatemofetil and steroids.Approximately 18 months after transplant, she presented with bilaterallower limb edema and significant proteinuria (7 gm/24 hrs) along withhypoalbuminemia and elevated serum creatinine (2.1 mg/dl). Renaltransplant biopsy demonstrated recurrent FSGS. In the following months,the renal function continued to deteriorate. The renal graft failedapproximately 28 months after the transplant due to recurrent FSGS andchronic transplant glomerulopathy. She was maintained on dialysis untilapproximately 3 years after the transplant when she received a secondliving-related donor kidney transplant, this time from her aunt. She hadpre-transplant plasmapheresis followed by daclizumab, methylprednisoloneand Thymoglobulin induction. She received a single dose of rituximabwithin 24 hours of transplant. Kidney transplant biopsies were obtainedin the operating room after the kidney was flushed (pre-perfusionbiopsy) with lactated ringer's solution (with added heparin,solu-medrol, sodium bicarbonate and lidocaine) and 2 hourspost-reperfusion of the kidney. An external stent (from renal pelvis,into bladder, exiting through the abdominal wall) was placed in thenewly transplanted kidney to differentiate urine from the secondtransplant from the previously transplanted kidney.

The newly transplanted kidney began to make urine immediately but spoturinalysis obtained from the new allograft immediately post operativelyshowed increased urinary protein to creatinine ratio. Her creatininestabilized by the 3rd day post transplant to between 1.2 and 1.5 mg/dl.There was no evidence of reperfusion injury, acute tubular necrosis,cellular rejection, vascular rejection or glomerulosclerosis on theformalin fixed post-reperfusion biopsy. However, there was effacement ofthe foot processes and enlargement of the podocytes noted on thepost-reperfusion biopsy examined by electron microscopy. Pre andpost-reperfusion renal biopsies were also examined byimmunofluorescencestudy of B7-1/CD80. Pre and post-reperfusion biopsieswere embedded in OCT. Ten mm-thick sections were fixed for 10 minutes incold acetone and blocked in 10% goat serum for 30 minutes. Slides wereincubated with a goat anti-human B7.1/CD80 antibody (15 g/ml, R&DSystems, Minneapolis, Minn.) and with a mouse monoclonal antibodyagainst the podocyte protein synaptopodin (Mundel et al., J Cell Biolvol. 139:193-204, 1997) (Byodesign International, 1:100), followed byexposure to Alexa-488 secondary donkey anti-goat and Alexa-647anti-mouse antibodies (dilution of 1:200, Invitrogen). An irrelevantantibody isotype was used as negative control. Image acquisition wasperformed with Leica SP5 inverted confocal microscope. Thepre-reperfusion staining was negative forB7-1/CD80; however, theimmunohistological studies showed positive staining with B7-1/CD80 inthe post-reperfusion biopsywith partial colocalization with synaptopodinutilized as a specific podocyte marker. Since immediate FSGS recurrencewas highly suspected, a course of 4 sessions of alternative dayplasmapheresis with simultaneous administration of 1 dose of Abatacept(500 mg) was started on postoperative day (POD) 3. After peaking at 4.6,the urinary protein to creatinine ratio decreased progressively.Correspondingly, the serum albumin and total protein levels increased tonormal values. She was discharged with tacrolimus, rapamycin (due tomycophenolate mofetil intolerance), irbesartan and eplerenoneon POD 8with a random urinary protein to creatinine ratio 0.8 mg/mg. The patientunderwent one more session of plasmapheresis in the outpatient clinic.Since then (1.5 years after cessation of plasmapheresis and abatacepttreatment) she remained stable with creatinine levels of 1.3 mg/dl, andmild proteinuria with a normal serum albumin.

Case Report 2

A 16-year-old white Hispanic female was diagnosed at the age of 18months with FSGS. She was treated for severe cardiomyopathy andsteroid-resistant nephrotic syndrome, which progressed to ESRD over thecourse of 7 years. She subsequently underwent native bilateralnephrectomy at the age of 8 years and the first kidney transplantationat the age of 9 years from her 41-year-old mother with Thymoglobulin anddaclizumab induction, and tacrolimus, mycophenolate and corticosteroidsmaintenance therapy. She developed massive proteinuria (urineprotein/creatinine ratio >40) 2 days after the transplantation requiringplasmapheresis and angiotensin receptor blocker therapy. She continuedto be nephrotic with gradual deterioration of renal function, especiallyafter she developed acute rejection at 13 months post-transplant; therenal transplant biopsy at that time revealed recurrent FSGS, IA acuteT-cell-mediated rejection, moderate interstitial fibrosis/tubularatrophy, and calcineurin-inhibitor toxicity. She eventually lost herfirst graft function 4.5 years after the transplantation and returned todialysis therapy. While waiting for the second kidney transplantation,she developed latent tuberculosis, which required a 9-month isoniazidtreatment. She was highly sensitized, but the degree of sensitizationfell spontaneously from PRA of 60% to 20%, and she received her secondkidney transplantation from a 34-year-old deceased donor. The transplantprocedure went uneventfully with an external ureteral stent placement todistinguish urine of the second transplant kidney from the firsttransplant. The patient received induction therapy with Thymoglobulin (1mg/kg×5 doses) and basiliximab (20 mg×2 doses), as well as a single doseof rituximab (375 mg/m²), and was maintained on tacrolimus, mycophenolicacid and corticosteroids. As depicted in FIGS. 4A-4C, the kidneyallograft presented immediate function; however, spot urine revealedsignificant proteinuria (protein/creatinine ratio 9-17), suggestingearly recurrence of FSGS. During the initial hospital stay, 6 sessionsof plasmapheresis were performed without significant improvement inurine protein levels. A single dose of abatacept (500 mg) wasadministered on POD 9. She was discharged on POD 10 with abovemaintenance immunosuppression and candesartan. She had 3 additionalsessions of plasmapheresis in the outpatient clinic. Subsequent toreceiving abatacept, there was a marked decreased in urine proteinlevels. Currently, at 3 months post-transplant, the patient has a serumcreatinine level of 0.7 mg/dl and a urine protein/creatinine ratio of1.3.

In these trials, podocyte foot process effacement and swelling was notedby EM in the post-reperfusion biopsy consistent with early recurrentFSGS. This is the first demonstration of expression of B7-1/CD80 onpodocytes in post-reperfusion biopsies in patients with post-transplantproteinuria associated with recurrent FSGS immediately post-transplant.The recurrent FSGS proteinuria was treated with abatacept combined withplasmapheresis, angiotensin converting enzyme and angiotensin IIreceptor blocker based on a clinical diagnosis of presumed recurrence.

The results of these trials suggest that the expression of B7-1/CD80 inpodocytes may be induced immediately post-reperfusion in patients athigh risk for recurrent FSGS. Both patients responded to a combinationof plasmapheresis and abatacept. In the second case, the reduction inproteinuria was temporally associated with the administration ofabatacept. Abatacept may be effective in reducing proteinuria andprogression of recurrent glomerulosclerosis. If additional studiesconfirm a critical role for B7-1/CD80 in the pathogenesis of earlyproteinuria following kidney transplant for FSGS, abatacept could beutilized for pre-emptive therapy to avoid the recurrence by virtue ofits direct effect on the podocyte.

Example 5—Modulation of SMPDL-3b Affects Podocyte Cholesterol/LipidContent

SMPDL-3b affects both cellular cholesterol (as shown in FIG. 5A) andsphingolipid (ASMase as shown in FIG. 5B). Therefore, modulation ofSMPDL-3b per se or of cellular lipid through other agents such ascyclodextrin derivatives may represent a new strategy to prevent andtreat proteinuric kidney disease through the modulation of cellularlipid content.

Example 6—Methods of Lowering Plasma Membrane and Cellular Cholesterolfor the Prevention, Treatment, Cure or Reversal of Renal-RelatedDisorders

Referring to FIG. 6A, the antiproteinuric effects of CD were tested inan established experimental model of LPS induced proteinuria. Three miceper group were utilized. Mice were left untreated (CTRL), or receivedone injection of 4000 mg/kg Hydroxypropyl-beta-cyclodextrin (CD), or twoinjections of 200 μg LPS 24 hours apart (LPS), or received 4000 mg/kg ofCD 1 hour prior to LPS treatment (LPS+CD). Urines were collected at 30hours after the first LPS injection and analyzed for albumin andcreatinine content by ELISA. Albuminuria was determined as the ratiobetween albumin and creatinine (μg/mg). It was found that cholesteroldepletion by intravenous injection of Hydroxypropyl-beta-cyclodextrin(CD) protects from Lipopolysaccharide (LPS) induced proteinuria in mice.

Referring to FIG. 6B, the antiproteinuric effects of CD as described inFIG. 6A were accompanied by a prevention of LPS-stimulated increase inMyD88 expression as determined by WB in isolated glomeruli, suggestingthat CD may preserve podocyte function and thus prevent proteinuria invivo.

OTHER EMBODIMENTS

Any improvement may be made in part or all of the assays, kits, andmethod steps. All references, including publications, patentapplications, and patents, cited herein are hereby incorporated byreference. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein, is intended to illuminate the invention anddoes not pose a limitation on the scope of the invention unlessotherwise claimed. Any statement herein as to the nature or benefits ofthe invention or of the preferred embodiments is not intended to belimiting, and the appended claims should not be deemed to be limited bysuch statements. More generally, no language in the specification shouldbe construed as indicating any non-claimed element as being essential tothe practice of the invention. Although the experiments described hereinpertain to diabetes and FSGS, the assays, method and kits describedherein can be applied to any glomerular disease or disorder. Thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contraindicated bycontext.

1-45. (canceled)
 46. A method of preventing or treating primary orsecondary proteinuric glomerular disorder or recurrent proteinuricglomerular disease in a subject comprising administering atherapeutically effective amount of a composition comprising acyclodextrin or a cyclodextrin derivative that restores physiologicallipid content in podocytes or physiological lipid-related proteincontent in podocytes to the subject, wherein the therapeuticallyeffective amount is effective for preventing apoptosis of podocytes,preventing disruption of podocyte cytoskeleton, preventing accumulationof cholesterol and/or lipids in podocytes, and preventing or treatingprimary or secondary proteinuric glomerular disorder or recurrentproteinuric or glomerular disease in the subject.
 47. The method ofclaim 46, wherein the primary or secondary proteinuric glomerulardisorder or recurrent proteinuric glomerular disease is diabeticnephropathy.
 48. The method of claim 46, wherein the cyclodextrin orcyclodextrin derivative that restores physiological lipid content inpodocytes or physiological lipid-related protein content in podocytes ishydroxypropyl β cyclodextrin (HPBCD).
 49. The method of claim 46,wherein the composition is administered to the subject at one or moretime points selected from the group consisting of prior to kidneytransplantation, during kidney transplantation, and subsequent to kidneytransplantation.
 50. The method of claim 46, wherein the compositionfurther comprises a pharmaceutically acceptable carrier.
 51. A method ofpreventing progression of FSGS, or recurrence of FSGS after kidneytransplantation, or treating FSGS in a subject having FSGS, the methodcomprising administering to the subject a composition comprising acyclodextrin or a cyclodextrin derivative that is capable of at leastone of increasing SMPDL-3b levels in the subject, restoring cytoskeletonrearrangements in the subject, and decreasing or preventing B7-1expression or activity in the subject, wherein the composition isadministered to the subject in an amount effective to prevent or treatFSGS in the subject.
 52. The method of claim 51, wherein the compositionis administered to the subject at one or more time points selected fromthe group consisting of prior to kidney transplantation, during kidneytransplantation, and subsequent to kidney transplantation.
 53. Themethod of claim 51, wherein the cyclodextrin or cyclodextrin derivativeis hydroxypropyl β cyclodextrin (HPBCD).
 54. The method of claim 51,wherein the composition further comprises a pharmaceutically acceptablecarrier.
 55. A method of preventing or treating a renal-related disorderin a subject comprising administering to the subject a compositioncomprising a cyclodextrin or a cyclodextrin derivative in an amounteffective for reducing at least one of plasma membrane cholesterol,plasma membrane lipids, cellular cholesterol, and cellular lipids in thesubject.
 56. The method of claim 55, wherein the renal-related diseaseis primary glomerulopathy, secondary glomerulopathy, or apost-transplant recurrence of a glomerular disease selected from thegroup consisting of focal segmental glomerulosclerosis (FSGS),membranous nephropathy, minimal change disease, IgA nephropathy,membranoproliferative glomerulopathy, diabetic nephropathy, lupusnephritis, myeloma kidney, hypertensive nephrosclerosis, paucimmuneglomerulonephritis, preeclampsia, amyloidosis, cryoglobulinemia,thrombotic thrombocytopenic purpura, hemolytic uremic syndrome,scleroderma kidney, Alport's glomerulopathy, transplant glomerulopathy,a storage disorder, stroke, peripheral vascular disease, diabetes,coronary artery disease, congestive heart failure, atherosclerosis,cardiac hypertrophy, myocardial infarction, endothelial dysfunction, andhypertension.
 57. The method of claim 56, wherein the composition isadministered in an amount effective for preserving podocyte function andpreventing or treating proteinuria in the subject.
 58. The method ofclaim 55, wherein administration of the composition results in reductionof at least one of: plasma membrane cholesterol, plasma membrane lipids,cellular cholesterol, and cellular lipids in podocytes of the kidney ofthe subject.
 59. The method of claim 55, wherein the composition furthercomprises a drug selected from the group consisting of animmunosuppressive agent, ACTH agonist, insulin sensitizer, GHantagonist, antinflammatory medication, vitamin D derivative, RAS systeminhibitor, aldosterone inhibitor, HMG-CoA reductase inhibitor,cholesterol absorption inhibitor, bile acid sequestrant, bile acidresin, niacin, niacin derivative, fibrate, cholesteryl ester transferprotein (CETP) inhibitor, Acetyl-Coenzyme A acetyltransferase (ACAT)inhibitor, and microsomal triglyceride transport inhibitor.
 60. Themethod of claim 55, wherein the composition is administered to thesubject at one or more time points selected from the group consisting ofprior to kidney transplantation, during kidney transplantation, andsubsequent to kidney transplantation.
 61. The method of claim 55,wherein the composition further comprises a pharmaceutically acceptablecarrier.
 62. The method of claim 55, wherein the cyclodextrin orcyclodextrin derivative is hydroxypropyl β cyclodextrin (HPBCD).